Engineered human T cell containing switchable chimeric antigen cell surface receptor and preparation method thereof

Abstract

The present invention relates to an engineered human T cell comprising a switchable chimeric antigen cell surface receptor, a pharmaceutical composition comprising the engineered human T cell, a kit comprising the engineered human T cell and a targeting module, and a method of preparing the engineered human T cell.

Description

Technical Field

[0001] This invention relates to an engineered human T cell comprising a switchable chimeric antigen cell surface receptor, a pharmaceutical composition comprising engineered human T cells, a kit comprising engineered human T cells and a targeting module, and a method for preparing engineered human T cells. Background Technology

[0002] Chimeric antigen receptors (CARs) are artificial receptors consisting of an antigen-specific binding moiety and one or more signaling chains derived from immune receptors (Cartellieri et al., 2010). Genetically modified immune cells expressing CARs can bind to cells or tissue structures expressing the corresponding target of the CAR binding moiety. Crosslinking can induce signaling pathways via the CAR signaling chain, which alters the biological characteristics of the CAR-transplanted immune cells. Conversely, CAR activation in genetically modified regulatory T cells (Tregs) can induce Treg-specific immunomodulatory and suppressive mechanisms, such as the secretion of interleukin (IL)-10 or tumor growth factor-β (TGF-β). Adoptive transfer of chimeric antigen receptor (CAR)-engineered immune cells is currently considered a very promising therapeutic option for treating previously incurable malignancies, infectious diseases, or autoimmune diseases.

[0003] However, conventional CAR technology faces several key challenges that need to be addressed before this treatment can be widely adopted in clinical practice. First, several safety issues must be resolved. To date, the immune response to T cells engineered with conventional CARs after infusion into patients has been difficult to control. The incidence of serious adverse events is high (Titov et al., 2018). In particular, unexpected target gene expression in normal tissues can trigger a rapid and intense immune response of engineered T cells against normal cells, potentially leading to serious side effects (Morgan et al., 2010). Furthermore, because CAR-T cells are a novel class of self-expanding cell therapies, in the presence of a heavy tumor burden, infused T cells can undergo dramatic expansion, leading to tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome (Brudno and Kochenderfer, 2016). Another drawback of conventional CAR technology is that engineered T cells can only retarget a single antigen. This single-target approach implies a risk of generating tumor escape variants that lose their target antigens during treatment. Tumor escape variants have been observed in clinical trials several months after conventional CAR T-cell therapy (Sotillo et al., 2015). In summary, these obstacles limit the application of CAR T-cells to a very limited number of indications. In fact, to date, most instances of clinical efficacy have been observed with CAR T-cells targeting CD19 and BCMA.

[0004] The modular, switchable “universal” CAR T (UniCAR) approach overcomes these limitations by separating the antigen recognition domain and activation domain of the CAR into two independent operational units. T cells are genetically engineered to express a CAR with a universal binding domain that recognizes a tag (Cartellieri et al., 2016). Antigen specificity is provided by soluble adaptor molecules, which consist of an antigen-binding domain fused to a tag recognized by the universal CAR. Cartellieri et al. described the therapeutic effects on CD33 and / or CD123-positive acute myeloid leukemia cells in vitro and in vivo.

[0005] In addition to the UniCAR approach (EP 2 990 416 A1) for recognizing various antigens, a reverse universal CAR (RevCAR) approach is also known, which facilitates the binding of genetically engineered immune cells expressing a tagged RevCAR to target cells via an adaptor molecule containing a tag-binding domain and a target cell-binding domain (EP 3 581 200 A1 and WO2019 / 238722A1). WO 2019 / 238722 A1 discloses a tag derived from extracellular LA / SSB and an adaptor molecule containing a CD123 scFv and an scFv that binds to the tag (5B9 or 7B6). Adding this adaptor molecule to link the CAR and tumor cells produces antigen-specific cytotoxicity.

[0006] Liu et al. described switchable and programmable universal CARs for CAR T-therapy. D3 reviews different approaches to engineering switchable CARs, including anti-5B9 UniCARs targeting a 5B9 tag on a bispecific switching molecule that targets cancer antigens such as CD123 or CD33 (Liu et al., 2019).

[0007] Furthermore, switchable CAR-T methods, such as UniCAR or RevCAR, allow CAR T cells to rest between activation and stimulation cycles by pausing the administration of soluble adaptor molecules. This holds promise for preventing exhaustion of conventional CAR T cells under continuous stimulation, thereby improving durability (Weber et al., 2021).

[0008] To avoid immune rejection, autologous methods such as UniCAR or RevCAR are used to genetically engineer immune cells, using the patient's own cells as the cell source for treatment. However, this method is time-consuming and costly, and patients have often received multiple treatments, which may adversely affect the functional characteristics of the immune cells.

[0009] To address this issue, allogeneic cell transplantation into the recipient could be considered. However, the use of allogeneic cells is limited because the recipient's immune cells may recognize the transplanted cells as foreign and attack them. Immune rejection of allogeneic cells is typically due to a mismatch between the major histocompatibility complex (MHC) molecules between the donor and recipient. In the human population, multiple genetic variants exist for any MHC gene, encoding different forms of MHC proteins. Human MHC class I molecules include HLA-A, HLA-B, and HLA-C molecules, which are expressed in all nucleated cells and platelets and present epitopes to activate cytotoxic T cells. MHC class II molecules include HLA-DP, HLA-DQ, and HLA-DR molecules and are expressed only on specific antigen-presenting cells (e.g., macrophages, B cells, and dendritic cells (DCs)) and present antigens to activate helper T cells, which in turn signal B cells to produce antibodies.

[0010] Subtle differences in MHC alleles between individuals can lead to the activation of recipient T cells after donor T cell transplantation. During T cell development, an individual's T cell pool develops tolerance to its own MHC molecules, but T cells that recognize the MHC molecules of another individual may continue to circulate and are known as allogeneic reactive T cells. Allogeneic reactive T cells can be activated by the presence of cells from another individual expressing MHC molecules in the body, leading to graft-versus-host disease and transplant rejection.

[0011] Methods and compositions for reducing the susceptibility of allogeneic cells to rejection have attracted considerable attention, including reducing the expression of MHC proteins in cells to avoid recipient T cell responses. In practice, the ability to genetically modify allogeneic cells for transplantation into subjects is hampered by the need for multiple gene edits to reduce the expression of all MHC proteins while simultaneously avoiding other harmful recipient immune responses. One approach utilizes prolonged immunosuppression to avoid immune rejection and improve persistence (Neelapu et al., 2020). While this approach has shown some clinical effectiveness, it increases the risk of infection, and the persistence of adoptive T cells is uncertain and dependent on sustained immunosuppression. Other strategies include knocking out the β2M gene to remove all HLA class I molecules (HLA-A, HLA-B, and HLA-C) from the surface of T cells to avoid recognition by host CD8+ T cells. However, the loss of HLA class I signals a “self-deletion” to host NK cells, which readily eliminate β2M KO T cells. To overcome this problem, researchers are exploring the overexpression of non-polymorphic HLA-E genes, which can provide partial protection but not complete protection against NK cell-mediated lysis (Zhang, 2021). Ellis et al. described T-cell genetic engineering for immunotherapy, specifically the genetic regulation of engineered T cells to improve safety and efficacy, particularly by genetically deleting the expression of HLA class I and HLA class II by targeting B2M and CIITA respectively, thereby prolonging the survival of chimeric antigen receptor (CAR) T cells in preclinical models (Ellis et al., 2021). However, it has been revealed that allogeneic CAR cells lacking HLA molecules are killed by the recipient's natural killer cells, which may require overexpression of HLA-E or other non-classical major histocompatibility complex (MHC) molecules as a remedy.

[0012] Qasim et al. described the engineering of T cells to express a chimeric antigen receptor against the B cell antigen CD19 (CAR19) by transducing non-human leukocyte antigen-matched donor cells with lentiviruses and simultaneously performing transcription activator effector nuclease (TALEN)-mediated gene editing on the T cell receptor α chain and CD52 gene locus (Qasim et al., 2017). Summary of the Invention

[0013] Therefore, the object of the present invention is to provide an engineered allogeneic immune cell comprising a switchable chimeric antigen cell surface receptor, wherein the engineered allogeneic immune cell can reduce the rejection response of the subject's immune cells and has excellent durability by combining the avoidance of rejection response with the avoidance of exhaustion caused by continuous stimulation.

[0014] The present invention achieves the above-mentioned objectives by engineered human T cells, a kit, and a method for preparing engineered human T cells according to the independent claims.

[0015] Preferred embodiments are described in the dependent claims of this invention.

[0016] A first aspect of the present invention provides an engineered human T cell, the engineered human T cell comprising:

[0017] i. By modifying the T cell receptor α chain gene, the surface expression of endogenous T cell receptor α chain (TRAC) can be reduced or eliminated;

[0018] ii. By modifying the HLA-A gene, the surface area of ​​HLA-A is reduced or eliminated compared to unmodified T cells.

[0019] Express;

[0020] iii. By modifying the CIITA gene, the surface expression of HLA class II can be reduced or eliminated, and

[0021] iv. Switchable chimeric antigen cell surface receptors, including:

[0022] Tags combined with fields or tags,

[0023] Extracellular hinges and transmembrane domains, and

[0024] Signal transduction structural domain.

[0025] Preferably, the engineered human T cells of the present invention, when used in combination with a targeting module that actively targets antigens expressed by tumor cells, can induce a significant anti-tumor response, and the anti-tumor response of the switchable chimeric antigen receptor is induced only in the presence of the targeting module. This effect can be reversibly interrupted by discontinuing the administration of the targeting module. More preferably, the targeting module has a short pharmacokinetic and pharmacodynamic half-life, providing a rapid and reversible shutdown mechanism for the mediated immune response. More preferably, the knockout of the three genes (TRAC, HLA-A gene, CIITA) of the present invention leads to a reduction in the rejection response of the subject's immune cells, particularly reducing the probability of allogeneic T cells developing graft-versus-host disease (GvHD).

[0026] In this invention, the term "domain" refers to a portion of a protein sequence that exists and functions independently of the rest of the protein.

[0027] In this invention, engineered human T cells reduce or eliminate the surface expression of endogenous T cell receptor α chain (TRAC) by modifying the TRAC gene, thereby preventing graft-versus-host disease.

[0028] In some embodiments, gene modification of the TRAC gene includes modifying at least one nucleotide in the genomic coordinates chr14:22547524-chr14:22547544. In some embodiments, gene modification of the TRAC gene includes modifying at least 10 or at least 15 consecutive nucleotides in the said genomic coordinates.

[0029] In some embodiments, genetic modification of the TRAC gene involves modifying at least one nucleotide of an exon of the TRAC gene.

[0030] In some embodiments, genetic modification of the TRAC gene includes modifying at least one nucleotide in the genomic coordinates by at least one of insertion, deletion, substitution, or deamination.

[0031] In some embodiments, the genetic modification of the TRAC gene includes insertion / deletion mutations (indels).

[0032] In some embodiments, TRAC gene expression is reduced or eliminated by a gene editing system that binds to a TRAC genomic target sequence comprising at least five consecutive nucleotides within a genomic coordinate range, preferably chr14:22547524-chr14:22547544.

[0033] In this invention, compared to unmodified T cells, engineered human T cells reduce or eliminate HLA-A surface expression by genetically modifying the HLA-A gene.

[0034] In some embodiments, modification of the HLA-A gene includes modifying at least one nucleotide of a genomic coordinate selected from the ranges of chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619.

[0035] In some embodiments, the T cells have a homozygous HLA-B genotype and / or a homozygous HLA-C genotype. In some embodiments, both the HLA-B and HLA-C genotypes of the T cells are homozygous.

[0036] In some embodiments, T cells reduce or eliminate the expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24.

[0037] In some embodiments, the genetic modification of the HLA-A gene includes modifying at least one nucleotide in genomic coordinates chr6:29942864-chr6:29942903, preferably at least one nucleotide in genomic coordinates chr6:29942876-chr6:29942897.

[0038] In some embodiments, the genetic modification of the HLA-A gene includes modifying at least one nucleotide in genomic coordinates chr6:29943528-chr6:29943609, preferably at least one nucleotide in genomic coordinates chr6:29943528-chr6:29943550.

[0039] In some embodiments, gene modification of the HLA-A gene includes modifying at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides in the genomic coordinates, preferably at least 10, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotides in the genomic coordinates.

[0040] In some embodiments, genetic modification of the HLA-A gene includes at least one C to T substitution or at least one A to G substitution in the genomic coordinates.

[0041] In some embodiments, the genetic modification of the HLA-A gene includes insertions / deletions.

[0042] In some embodiments, HLA-A expression is reduced or eliminated by a gene-editing system that binds to an HLA-A genomic target sequence comprising at least five consecutive nucleotides within a genomic coordinate range, preferably within the following genomic coordinate ranges: chr6:29942864-29942884, chr6:29942868-29942888, chr6:29942876-29942896, chr6:29942877-29942897, chr6:29942883. -29942903, chr6:29943126-29943146, chr6:29943528-29943548, chr6:29943529-29943549, chr6:29943530-29943550, chr6:29943537-29943557, chr6:29943549-29943569, chr6:29943589-29943609 and chr6:29944026-29944046.

[0043] In certain embodiments, the HLA-B allele is selected from: HLA-B*07:02, HLA-B*08:01, HLA-B*44:02, HLA-B*35:01, HLA-B*40:01, HLA-B*57:01, HLA-B*14:02, HLA-B*15:01, HLA-B*13:02, HLA-B*44:03, HLA-B*38:01, HLA-B*18:01, HLA-B*44:03, HLA-B*51:01, HLA-B*49:01, HLA-B*15:01, HLA-B*18:01, HLA-B*27:05, HLA-B*35:03, HLA-B*18:01, HLA-B*52:01, HLA-B*51:01, HLA-B*37:01, HLA-B*53:01, HLA-B*55:01, HLA-B*44:02, HLA-B*44:03, HLA-B*35:02, HLA-B*15:01, and HLA-B*40:02.

[0044] In certain embodiments, the HLA-C allele is selected from: HLA-C*07:02, HLA-C*07:01, HLA-C*05:01, HLA-C*04:01, HLA-C*03:04, HLA-C*06:02, HLA-C*08:02, HLA-C*03:03, HLA-C*06:02, HLA-C*16:01, HLA-C*12:03, HLA-C*07:01, HLA-C*04:01, HLA-C*15:02, HLA-C*07:01, HLA-C*03:04, HLA-C*12:03, HLA-C*02:02, HLA-C*04:01, HLA-C*05:01, HLA-C*12:02, HLA-C*14:02, HLA-C*06:02, HLA-C*04:01, HLA-C*03:03, HLA-C*07:04, HLA-C*07:01, HLA-C*04:01, HLA-C*04:01, and HLA-C*02:02.

[0045] In certain embodiments, the HLA-B allele is selected from: HLA-B*07:02, HLA-B*08:01, HLA-B*44:02, HLA-B*35:01, HLA-B*40:01, HLA-B*57:01, HLA-B*14:02, HLA-B*15:01, HLA-B*13:02, HLA-B*44:03, HLA-B*38:01, HLA-B*18:01, HLA-B*44:03, HLA-B*51:01, HLA-B*49:01, HLA-B*15:01, HLA-B*18:01, HLA-B*27:05, HLA-B*35:03, HLA-B*18:01, HLA-B*52:01, HLA-B*51:01, HLA-B*37:01, HLA-B*53:01, HLA-B*55:01, HLA-B*44:02, HLA-B*44:03, HLA-B*35:02, HLA-B*15:01, and HLA-B*40:02, and the HLA-C allele is selected from: HLA-C*07:02, HLA-C*07:01, HLA-C*05:01, HLA-C*04:01, HLA-C*03:04, HLA-C*06:02, HLA-C*08:02, HLA-C*03:03, HLA-C*06:02, HLA-C*16:01, HLA-C*12:03, HLA-C*07:01, HLA-C*04:01, HLA-C*15:02, HLA-C*07:01, HLA-C*03:04, HLA-C*12:03, HLA-C*02:02, HLA-C*04:01, HLA-C*05:01, HLA-C*12:02, HLA-C*14:02, HLA-C*06:02, HLA-C*04:01, HLA-C*03:03, HLA-C*07:04, HLA-C*07:01, HLA-C*04:01, HLA-C*04:01, and HLA-C*02:02.

[0046] In some embodiments, the HLA-B and HLA-C alleles are selected from any of the following combinations: HLA-B*07:02 and HLA-C*07:02, HLA-B*08:01 and HLA-C*07:01, HLA-B*44:02 and HLA-C*05:01, HLA-B*35:01 and HLA-C*04:01, HLA-B*40:01 and HLA-C*03:04, HLA-B*57:01 and HLA-C*06:02, HLA-B*14:02 and HLA-C*06:02, HLA-B*14:02 and HLA-C*07:02, HLA-B*08:01 and HLA-C*07:01, HLA-B*57:01 and HLA-C*06:02, HLA-B*14:02 and HLA-C*07:02, HLA-B*08:01 and HLA-C*07:01 ... 2 and HLA-C*08:02, HLA-B*15:01 and HLA-C*03:03, HLA-B*13:02 and HLA-C*06:02, HLA-B*44:03 and HLA-C*16:01, HLA-B*38 :01 and HLA-C*12:03, HLA-B*18:01 and HLA-C*07:01, HLA-B*44:03 and HLA-C*04:01, HLA-B*51:01 and HLA-C*15:02, HLA-B*4 9:01 and HLA-C*07:01, HLA-B*15:01 and HLA-C*03:04, HLA-B*18:01 and HLA-C*12:03, HLA-B*27:05 and HLA-C*02:02, HLA-B *35:03 and HLA-C*04:01, HLA-B*18:01 and HLA-C*05:01, HLA-B*52:01 and HLA-C*12:02, HLA-B*51:01 and HLA-C*14:02, HLA- B*37:01 and HLA-C*06:02, HLA-B*53:01 and HLA-C*04:01, HLA-B*55:01 and HLA-C*03:03, HLA-B*44:02 and HLA-C*07:04, HL A-B*44:03 and HLA-C*07:01, HLA-B*35:02 and HLA-C*04:01, HLA-B*15:01 and HLA-C*04:01, and HLA-B*40:02 and HLA-C*02:02.

[0047] In some embodiments, compared to unmodified T cells, the T cells further reduce or eliminate HLA-B surface expression through genetic modification of the HLA-B gene, and / or reduce or eliminate HLA-C surface expression through genetic modification of the HLA-C gene.

[0048] In some embodiments, compared to unmodified T cells, the T cells also have reduced or eliminated HLA-B surface expression through genetic modification of the HLA-B gene, and the HLA-C genotype of the T cells is homozygous.

[0049] In an alternative embodiment, compared to unmodified T cells, the T cells also reduce or eliminate HLA-C surface expression through genetic modification of the HLA-C gene, and the HLA-B genotype of the T cells is homozygous.

[0050] In other alternative embodiments, the T cells reduce or eliminate HLA-B surface expression by genetic modification of the HLA-B gene and reduce or eliminate HLA-C surface expression by genetic modification of the HLA-C gene, compared to unmodified T cells.

[0051] In this invention, engineered human T cells contain HLA class II surface expression reduced or eliminated by genetic modification of the CIITA gene.

[0052] In some embodiments, genetic modification of the CIITA gene includes modifying at least one nucleotide at a splice site in genomic coordinates chr16:10902171-chr16:10923242.

[0053] In some embodiments, genetic modification of the CIITA gene includes modification of at least one nucleotide at the splice acceptor site, wherein the nucleotide is A, G, or T.

[0054] In some embodiments, genetic modification of the CIITA gene includes modification of the nucleotides at the splice site boundaries.

[0055] In some embodiments, genetic modification of the CIITA gene involves modifying at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides at genomic coordinates chr16:10902171-chr16:10923242.

[0056] In some embodiments, the genetic modification of the CIITA gene includes at least one C to T substitution or at least one A to G substitution in genomic coordinates chr16:10902171-chr16:10923242.

[0057] In some embodiments, genetic modifications to the CIITA gene include insertion / deletion mutations, C-to-T substitutions, or A-to-G substitutions occurring within a genomic coordinate range selected from one of the following ranges: chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr1 6:10902183-10902203, chr16:10902184-10902204, chr16:10902644-10 902664, chr16:10902779-10902799, chr16:10902788-10902808, chr16:1 0902789-10902809, chr16:10902790-10902810, chr16:10902795-109028 15. chr16:10902799-10902819, chr16:10903708-10903728, chr16:10903 713-10903733、chr16:10903718-10903738、chr16:10903721-10903741、 chr16:10903723-10903743, chr16:10903724-10903744, chr16:10903873 -10903893、chr16:10903878-10903898、chr16:10903905-10903925、chr 16:10903906-10903926, chr16:10904736-10904756, chr16:10904790-10 904810、chr16:10904811-10904831、chr16:10906481-10906501、chr16: 10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906 507、chr16:10906492-10906512、chr16:10908127-10908147、chr16:1090 8130-10908150, chr16:10908131-10908151, chr16:10908132-10908152,chr16:10908137-10908157、chr16:10908138-10908158、chr16:10908139-10908159、chr16:10909006-10909026、chr16:10909007-109090 27. chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-109 10185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552- 10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:109164 49-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:109 18504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16: 10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242.

[0058] In some embodiments, genetic modification of the CIITA gene comprises modifying at least five consecutive nucleotides selected from the following genomic coordinates: chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152, chr 16:10908137-10908157, chr16:10908138-10908158, chr16:10908139-1 0908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16: 10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909 042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:1091 0176-10910196、chr16:10910186-10910206、chr16:10915547-10915567 , chr16:10915551-10915571, chr16:10915552-10915572, chr16:1091556 7-10915587、chr16:10916348-10916368、chr16:10916359-10916379、chr 16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-10 916470、chr16:10916455-10916475、chr16:10916456-10916476、chr16: 10918423-109l8443、chr16:10918504-10918524、chr16:10918511-10918 531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:1092 2153-10922173、chr16:10922478-10922498、chr16:10922487-10922507、chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241 and chr16: 10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:1091 8511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, c hr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10 922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430 and chr16:10908130-10908150.

[0059] In some embodiments, genetic modification of the CIITA gene involves modifying at least 10 or at least 15 consecutive nucleotides within the genomic coordinate range.

[0060] In some embodiments, the genetic modification of the CIITA gene includes insertion / deletion mutations.

[0061] In some embodiments, genetic modification of the CIITA gene includes modifying at least one nucleotide at a splice site in genomic coordinates chr16:10902171-chr16:10923242.

[0062] In some embodiments, genetic modification of the CIITA gene involves modifying at least one nucleotide at the splice acceptor site, wherein the nucleotide is A, G, or T.

[0063] In some embodiments, genetic modification of the CIITA gene includes modification of the nucleotides at the splice site boundaries.

[0064] In some embodiments, the genetic modification of the CIITA gene comprises at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides in genomic coordinates chr16:10902171-chr16:10923242.

[0065] In some embodiments, the genetic modification of the CIITA gene includes at least one C to T substitution or at least one A to G substitution in genomic coordinates chr16:10902171-chr16:10923242.

[0066] In some embodiments, genetic modifications to the CIITA gene include insertion / deletion mutations, C-to-T substitutions, or A-to-G substitutions occurring within a genomic coordinate range selected from one of the following ranges: chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr16 :10902183-10902203, chr16:10902184-10902204, chr16:10902644-109 02664, chr16:10902779-10902799, chr16:10902788-10902808, chr16:10 902789-10902809, chr16:10902790-10902810, chr16:10902795-109028 15. chr16:10902799-10902819, chr16:10903708-10903728, chr16:10903 713-10903733、chr16:10903718-10903738、chr16:10903721-10903741、 chr16:10903723-10903743, chr16:10903724-10903744, chr16:10903873 -10903893、chr16:10903878-10903898、chr16:10903905-10903925、chr 16:10903906-10903926, chr16:10904736-10904756, chr16:10904790-10 904810、chr16:10904811-10904831、chr16:10906481-10906501、chr16: 10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906 507、chr16:10906492-10906512、chr16:10908127-10908147、chr16:1090 8130-10908150, chr16:10908131-10908151, chr16:10908132-10908152,chr16:10908137-10908157、chr16:10908138-10908158、chr16:10908139-10908159、chr16:10909006-10909026、chr16:10909007-109090 27. chr16:10909018-10909038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-109 10185, chr16:10910176-10910196, chr16:10910186-10910206, chr16:10915547-10915567, chr16:10915551-10915571, chr16:10915552- 10915572, chr16:10915567-10915587, chr16:10916348-10916368, chr16:10916359-10916379, chr16:10916362-10916382, chr16:109164 49-10916469, chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:109 18504-10918524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16: 10922478-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242.

[0067] In some embodiments, genetic modification of the CIITA gene comprises modifying at least five consecutive nucleotides selected from the following genomic coordinate ranges: chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152 ... r16:10908137-10908157, chr16:10908138-10908158, chr16:10908139- 10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16 :10909018-10909038, chr16:10909021-10909041, chr16:10909022-1090 9042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:109 10176-10910196、chr16:10910186-10910206、chr16:10915547-1091556 7. chr16:10915551-10915571, chr16:10915552-10915572, chr16:109155 67-10915587, chr16:10916348-10916368, chr16:10916359-10916379, ch r16:10916362-10916382, chr16:10916449-10916469, chr16:10916450-1 0916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16: 10918423-109l8443、chr16:10918504-10918524、chr16:10918511-10918 531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:1092 2153-10922173、chr16:10922478-10922498、chr16:10922487-10922507、chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241 and chr16: 10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:1091 8511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, c hr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10 922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430 and chr16:10908130-10908150.

[0068] In some embodiments, genetic modification of the CIITA gene involves modifying at least 10 or at least 15 consecutive nucleotides within the genomic coordinate range.

[0069] In this invention, engineered human T cells contain switchable chimeric antigen cell surface receptors, which include a tag-binding domain or tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain.

[0070] The term "switchable chimeric antigen receptor" refers to an artificial chimeric fusion protein, specifically a receptor comprising a tag-binding domain or tag, an extracellular hinge and transmembrane domain, and a signal transduction domain. Figure 1The aforementioned domains can be derived from different sources, hence the term chimeric. Receptors can bind to different target modules via tag-binding domains or tags.

[0071] In some embodiments, engineered human T cells contain an exogenous nucleotide sequence encoding a switchable CAR, which is expressed on the surface of the engineered T cells.

[0072] Engineered human T cells containing a nucleotide sequence encoding a switchable CAR express the switchable CAR, which has binding specificity to the tag-binding domain or tag of the targeting module, which in turn binds to an antigen on the target cell.

[0073] In some embodiments, the switchable chimeric antigen cell surface receptor is a reverse universal chimeric antigen cell surface receptor that includes a tag, an extracellular hinge and a transmembrane domain, and a signal transduction domain.

[0074] Engineered human T cells containing reverse universal chimeric antigen cell surface receptors are less exhausted after generation and exhibit a less differentiated phenotype.

[0075] The term "tag" refers to a marker, such as a peptide sequence or organic molecule, that is attached to a peptide or protein so that it can bind to specific atoms, ions, or molecules (especially tag-binding domains).

[0076] In some embodiments, the tag is an organic molecule, including fluorescent markers such as FITC (fluorescein isothiocyanate) and biotin.

[0077] In some embodiments, the tag is a peptide epitope tag. In some embodiments, the tag contains 10 to 20 amino acids.

[0078] In some embodiments, the peptide epitope tag is a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, preferably a short linear peptide sequence or a mutant thereof as shown in SEQ ID No. 7 or SEQ ID No. 8; a leucine zipper sequence, preferably SYNZIP 1 to SYNZIP 48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), such as the sequence shown in SEQ ID No. 9 or SEQ ID No. 10 or a mutant thereof; or a short linear peptide sequence from human nucleoprotein, preferably from human La protein, more preferably a short linear peptide sequence or a mutant thereof as shown in SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13.

[0079] The term "nuclear protein" refers to proteins found in the cell nucleus. Because the tag is a peptide sequence derived from a nuclear antigen, it cannot be recognized and bound by the corresponding tag-binding domain under physiological conditions and in the environment of native proteins. Furthermore, the tag is non-immunogenic, thus minimizing the risk of uncontrolled extra-site targeting toxicity from CAR-expressing immune cells, such as the release of toxic levels of cytokines, also known as a cytokine storm or cytokine release syndrome (CRS).

[0080] In some embodiments, the His-tag is an amino acid sequence consisting of histidine residues, preferably in the range of 6 to 14.

[0081] In some embodiments, the peptide epitope tag is a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, preferably a short linear peptide sequence as shown in SEQ ID No. 7 or SEQ ID No. 8; a leucine zipper sequence, preferably SYNZIP 1 to SYNZIP 48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), or a sequence as shown in SEQ ID No. 9 or SEQ ID No. 10; or a short linear peptide sequence from human nucleoprotein, preferably from human La protein, more preferably a short linear peptide sequence as shown in SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13.

[0082] Preferably, the peptide epitope tag from human La protein is human La epitope E5B9 as shown in SEQ ID No. 11 or E7B6 as shown in SEQ ID No. 12 or SEQ ID No. 13, and most preferably human La epitope E5B9 as shown in SEQ ID No. 11 or E7B6 as shown in SEQ ID No. 13.

[0083] The term "mutant" refers to a peptide or protein that has at least 90% sequence identity, preferably at least 95%, with a specified peptide or protein. A mutant can possess one or more activities of the specified peptide or protein, particularly the ability to bind the same tag-binding domain to a peptide epitope tag.

[0084] In some embodiments, the mutant is a truncated form of a peptide or protein. The term "truncated form" refers to a shortened peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 90% chain length and 100% sequence identity, and most preferably at least 95% chain length and 100% sequence identity. The activity of the truncated form is at least 90%, preferably at least 95%, of the activity of the specified peptide or protein.

[0085] In some embodiments, the tag-binding domain is an antibody, antibody fragment, protein, or peptide.

[0086] The term "antibody" refers to a protein that binds to an antigen via the variable region (Fab) of its antigen-binding fragment. Antibodies consist of a heavy chain (V... H ) and light chains (V L Each consists of a constant domain and a variable domain. The term "antibody fragment or antigen-binding fragment" refers to a fragment containing at least an antibody. L or V H Proteins. In some embodiments, the antibody fragment is selected from single-chain variable fragments (scFv), single-chain antibodies, F(ab')2 fragments, Fab fragments, and fragments or single-domain antibodies (nanobodies) generated from Fab expression libraries.

[0087] The term "single-chain variable fragment (scFv)" refers to a synthetically produced antibody fragment comprising a variable region of the antibody light chain and a variable region of the heavy chain linked by covalent bonds. In some embodiments, the V of the antibody... L and V H Short peptides of 10 to 25 amino acids are linked by covalent bonds. In some embodiments, the short peptides will... H The N-terminus and V L The C-end connection, or vice versa.

[0088] Preferably, V H and V L via having structure (G x S y The linker is a glycine-serine linker, wherein x and y range from 1 to 10, preferably from 1 to 5. Most preferably, it consists of 1 to 10 repeating sequences of sequence G4S1 (SEQ ID No. 4). Furthermore, the linker is preferably composed of a peptide sequence capable of increasing the protease resistance of the antibody derivative. The term "derivative" refers to a molecule that has a high structural similarity to a given molecule, preferably a molecule with the same backbone, wherein at least one atom, group, functional group, or substructure is substituted by another atom, group, functional group, or substructure (e.g., a hydroxyl group). The derivative is capable of possessing one or more activities of the specified molecule.

[0089] In some embodiments, the sequence of the connector is as shown in SEQ ID No. 5 or SEQ ID No. 6.

[0090] In some embodiments, the tag-binding domain is an antibody or antibody fragment, protein, or peptide that may bind to the following tags: myc-tag, His-tag, short linear peptide sequence from yeast transcription factor GCN4, leucine zipper sequence, or short linear peptide sequence from human nuclear protein, preferably from human La protein.

[0091] In some embodiments, the tag-binding domain is an antibody or antibody fragment.

[0092] In some embodiments, the antibody is derived from an animal species, preferably a mammal, such as human, ape, mouse, rat, rabbit, guinea pig, horse, cattle, sheep, goat, pig, dog, or cat. Preferably, the antibody or antibody fragment is a human, humanized, or deimmunogenic antibody. Humanized antibodies can be prepared by various methods, for example, through surface reconstruction and CDR transplantation. In the case of surface reconstruction, molecular modeling, statistical analysis, and mutagenesis techniques are used in combination to modify all non-CDR regions on the antibody surface to make it similar to the antibody surface of the target organism. In CDR transplantation, the CDR regions of the present invention are introduced into known human framework regions, the sequences of which are similar to the original sequences. Deimmunogenic antibodies can be obtained by specifically mutagenizing residues that confer immunogenic hotspots, which are predicted based on computer-simulated peptide-major histocompatibility complex (MHC) affinity.

[0093] The term "CDR (Complementarity-Determining Region)" refers to the variable chain portion of an antibody or antibody fragment that binds to its specific antigen. An antibody contains three non-contiguous CDRs (CDR1, CDR2, and CDR3) along the amino acid sequence of each variable region, thus creating a region between the two variable domains (V... H and V L There are six CDRs on it, which can come into contact with the antigen.

[0094] In some embodiments, the antibody or antibody fragment is a polyclonal, monoclonal, or chimeric antibody, wherein the antigen-binding region of a non-human antibody is transferred into the framework of a human antibody using computer-designed recombinant DNA technology.

[0095] In some embodiments, antibodies against a selected tag or antigen may be generated by inducing an immune response in a host, including (but not limited to) goats, rabbits, rats, mice, and humans, by means of injecting the host with cells expressing a specific protein, DNA or RNA encoding the protein, the protein itself, or any portion, fragment, or oligopeptide that retains the immunogenicity of the protein.

[0096] In some embodiments, the tag-binding domain binds to a tag derived from a human nuclear La protein; preferably, the tag-binding domain is an antibody or antigen-binding fragment comprising a V sequence having the following... L :

[0097] DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTRX 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVEIK (SEQ ID No. 14), where X 24 X 35 X 61 and X 101 They are independently selected from proteinogenic α-amino acid residues;

[0098] or the V L The sequence has at least 90% sequence identity, preferably at least 95% sequence identity, with respect to sequences such as SEQ ID No. 16 or SEQ ID No. 18.

[0099] In some embodiments, the tag binding domain constitutes an anti-La epitope scFv.

[0100] In some embodiments, X 24 To X 101 The options are as follows:

[0101] X 24 Selected from polar and / or positively charged residues, such as serine, threonine, asparagine, glutamine, histidine, lysine, and arginine; preferably lysine or arginine;

[0102] X 35 Selected from lysine and proline;

[0103] X 61 It is selected from polar and charged residues, such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine;

[0104] X 101 It is selected from hydrophobic residues, such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline, and tryptophan; preferably leucine or proline.

[0105] In some embodiments, the tag-binding domain is an antibody or antigen-binding fragment containing a sequence having at least 90% sequence identity with sequence SEQ ID No. 15 or SEQ ID No. 17, preferably at least 95% sequence identity.

[0106] In some embodiments, the tag binding field includes elements such as SEQ ID NO.15 (V H ) and SEQ ID NO.16 (V L The sequence shown in SEQ ID NO.17(V) H ) and SEQ ID NO.18 (V L The sequence shown is a sequence with at least 90% sequence identity, preferably at least 95% sequence identity.

[0107] In some embodiments, the tag binding field includes a field having, for example, SEQ ID NO.15 (V H ) and SEQ ID NO.16 (V L Anti-La 5B9 scFv of the sequence shown in SEQ ID NO.17, or having the following characteristics: H ) and SEQ ID NO.18 (V L The sequence shown is anti-La 7B6 scFv.

[0108] The term “extracellular hinge and transmembrane domain” refers to a flexible peptide sequence linked to a tag-binding domain tag that anchors a switchable CAR to the cell membrane and protrudes from the cell surface for optimal binding to its specific target module.

[0109] In some embodiments, the extracellular hinge and transmembrane domain are selected from the following hinges and transmembrane domains: human CD28 molecule, CD8a chain, NK cell receptor (preferably natural killer group NKG2D), or portions of the antibody constant region, and combinations thereof. The term "combinations thereof" refers to a combination of different hinges and transmembrane domains.

[0110] For example, a combination of extracellular hinge and transmembrane domain is (but not limited to) the CD28 extracellular hinge and transmembrane domain, the CD8α extracellular hinge and transmembrane domain, or the combination of the IgG1 or IgG4 constant region with the CD28α, CD28, or CD137 transmembrane domain.

[0111] In some embodiments, the extracellular hinge and transmembrane domain comprise the extracellular domain of CD28, more preferably the extracellular domain in which the B7 binding site is mutated. Mutation at the B7 binding site results in loss of ligand binding ability. In some embodiments, the extracellular hinge and transmembrane domain comprise the CD28 transmembrane domain.

[0112] The term "signal transduction domain" refers to a peptide sequence that transmits signals into the cell through cross-linking between cells expressing switchable CARs (effect cells) and human cell surface proteins or protein complexes (target cells). The cross-linking between effector cells and target cells is mediated by the targeting module of this invention.

[0113] In some embodiments, the signal transduction domain is selected from the following domains: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), the cytoplasmic regions of DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptor, mutants thereof, and combinations thereof.

[0114] The term "mutant" refers to a protein that has at least 90% sequence identity, preferably at least 95%, with a signal transduction domain. Mutants transmit signals into cells in the same manner as the designated signal transduction domain by cross-linking cells expressing switchable CARs (effector cells) with human cell surface proteins or protein complexes (target cells).

[0115] In some embodiments, the mutant is a truncated form. The term "truncated form" refers to a shortened protein having at least 90% sequence identity with the signal transduction domain, preferably at least 95% sequence identity, more preferably at least 90% chain length and 100% sequence identity, and most preferably at least 95% chain length and 100% sequence identity. The activity of the truncated form is at least 90%, more preferably at least 95%, of the activity of the specified signal transduction domain.

[0116] Guedan et al. described the use of mutants of the cytoplasmic region of CD28 as signal transduction domains (Guedan et al., 2020).

[0117] In some embodiments, the signal transduction domains are selected from the following domains: cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), cytoplasmic region of CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), and CD360 (interleukin-21 receptor) activated Fc receptors.

[0118] In some embodiments, the signal transduction domain includes a CD28 intracellular domain, more preferably a CD28 intracellular domain in which the internalization motif is mutated and fused with the CD3ζ intracellular domain.

[0119] In some embodiments, the switchable chimeric antigen receptor includes at least one signal transduction domain, preferably two, three, four or more signal transduction domains, particularly preferably selected from: the cytoplasmic regions of CD28, CD137, CD134, CD278, DAP10 and CD27, the cytoplasmic regions of PD-1, CTLA-4, and the CD3 chain, DAP12, CD122, CD132, CD127, CD360, activated Fc receptors, mutants thereof, and combinations thereof.

[0120] In some embodiments, the switchable chimeric antigen receptor includes at least one signal transduction domain, preferably two, three, four or more signal transduction domains, particularly preferably selected from: cytoplasmic regions of CD28, CD137, CD134, CD278, DAP10 and CD27, cytoplasmic regions of PD-1, CTLA-4, CD3 chain, DAP12, CD122, CD132, CD127, CD360 and activated Fc receptor.

[0121] In some embodiments, the switchable chimeric antigen receptor includes an additional domain, which is a short peptide linker of the extracellular portion of the receptor, that can be used to detect chimeric antigen receptors on the cell surface or to stimulate chimeric antigen receptor T cells.

[0122] In some embodiments, the other domain forms a linear epitope for specific binding of a monoclonal antibody (mab) to the other domain. In some embodiments, the other domain comprises at least one linear epitope, preferably the E7B6 epitope of the sequence shown in SEQ ID No. 12 or SEQ ID No. 13.

[0123] In some embodiments, another domain is located between the tag-binding domain (or the tag) and the extracellular hinge domain (or a component of the extracellular hinge domain).

[0124] In vitro or in vivo, switchable CAR transplanted cells with an additional domain can be specifically stimulated to proliferate preferentially and persist for a longer period compared to untransplanted cells. Furthermore, the additional domain can also be used to purify switchable CAR transplanted cells from mixed cell populations, suppress switchable CAR transplanted cell-mediated immune responses, and eliminate switchable CAR transplanted cells in vivo.

[0125] In some embodiments, the switchable CAR includes a signal peptide. The signal peptide can be expressed on the cell surface of effector cells. In some embodiments, the signal peptide is located at the N-terminus of the switchable CAR nucleotide sequence preceding the tag-binding domain or tag. In some embodiments, the signal peptide targets the protein to a secretory pathway during or after co-translation, and the signal peptide is selected from heavy or light chain leader peptides such as CD28, CD8α, IL-2, lysozyme C, or human antibodies to avoid immunogenic reactions.

[0126] In some embodiments, the tag-binding domain or tag site is located at the amino terminus of the polypeptide containing the switchable CAR. Positioning the tag-binding domain or tag at the amino terminus allows the tag-binding domain or tag to have unimpeded access to the targeting module that binds to the target cell.

[0127] In some embodiments, the switchable CAR includes one of the sequences shown in SEQ ID No. 33 to SEQ ID No. 36. Preferably, the switchable CAR has any of the sequences shown in SEQ ID No. 33 to SEQ ID No. 36.

[0128] In some embodiments, the switchable CAR comprises a sequence as shown in SEQ ID No. 33. More preferably, the switchable CAR has a sequence as shown in SEQ ID No. 33.

[0129] In some embodiments, the method of using engineered human T cells to stimulate a chimeric antigen receptor-mediated immune response in mammals is preferably used to treat cancer, infectious diseases, or autoimmune diseases.

[0130] The term "autoimmune disease" refers to an abnormal immune response (autoimmunity) of the body to substances and tissues that are normally present in the body.

[0131] In some embodiments, engineered human T cells are shared with a targeting module or a nucleic acid, vector, or cell encoding the targeting module, the targeting module comprising a tag-binding domain or a tag and at least one target cell binding domain, wherein the tag-binding domain of the targeting module binds to a tag of a switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of a switchable chimeric antigen receptor, wherein the engineered human T cells and the targeting module are administered in combination.

[0132] The term "combination administration" refers to a therapeutic approach in which a targeted module is administered before, simultaneously with, and / or after administration of a vector or cell containing a nucleotide sequence encoding a switchable chimeric antigen receptor.

[0133] In some embodiments, the targeting module is administered 1 hour to 2 days, preferably 4 to 24 hours, prior to the administration of engineered human T cells. Administering the targeting module prior to the administration of engineered human T cells activates the switchable chimeric antigen receptor and promotes T cell proliferation, leading to their accumulation at the target site.

[0134] In some embodiments, engineered human T cells are administered simultaneously with the targeting module.

[0135] In some embodiments, the targeting module is preferably administered 3 to 30 days after the administration of engineered human T cells. Additionally, an extra dose of the targeting module can be administered after the resting period to reactivate effector cells carrying switchable CARs.

[0136] Another aspect of the present invention provides a pharmaceutical composition comprising engineered human T cells as described herein and a pharmaceutically acceptable diluent or carrier.

[0137] The pharmaceutical composition is preferably administered parenterally, and particularly preferably intravenously. In some embodiments, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion, or suspension.

[0138] In some embodiments, the pharmaceutical composition comprises a concentration of 1×10 5 Up to 1×10 8 Injectable buffer solutions for engineered human T cells in the range of / mL.

[0139] The pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In some embodiments, the carrier is selected from water, an aqueous buffer solution, 0.9% physiological saline, 5% glucose, 5% xylitol, 0.3% glycine solution, Ringer's solution, or an amino acid solution. In some embodiments, the aqueous buffer solution is selected from an aqueous solution of histidine, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate buffer solution with a pH in the range of 5.0 to 7.0. In some embodiments, the buffer concentration of the aqueous buffer solution is in the range of 1 mmol / L (mM) to 500 mM, preferably in the range of 5 mM to 20 mM, and particularly preferably in the range of 5 mM to 10 mM.

[0140] In some embodiments, the carrier comprises sodium chloride, preferably in the range of 1 mM to 300 mM, particularly preferably 150 mM.

[0141] In some embodiments, the pharmaceutical composition further comprises a stabilizer, preferably in the range of 1 mM to 900 mM, particularly preferably in the range of 50 mM to 600 mM. In some embodiments, the stabilizer is sucrose, trehalose, or L-methionine.

[0142] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a compound that provides a near-physiological state and / or increases stability, such as a pH adjuster, buffer, and toxicity modifier. In some embodiments, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and polysorbate-80, preferably in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v) of polysorbate-80.

[0143] In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized using conventional, well-known techniques, including (but not limited to) sterile filtration.

[0144] In some embodiments, the pharmaceutical composition is used to administer the drug composition to a subject.

[0145] In some embodiments, the pharmaceutical composition is lyophilized before storage, or stored in solution form at room temperature or lower, including but not limited to frozen storage.

[0146] In some embodiments, the pharmaceutical composition is reconstituted and / or diluted in an infusion / stabilizer solution prior to administration to a subject. The solution used for reconstitution or the infusion / stabilizer solution may contain any of the components or similar components mentioned in the pharmaceutical composition.

[0147] In some embodiments, the pharmaceutical composition is used in a method of stimulating a chimeric antigen receptor-mediated immune response in mammals, preferably for treating cancer, infectious diseases, or autoimmune diseases.

[0148] In some embodiments, the pharmaceutical composition is used to treat cancer, infectious diseases, or autoimmune diseases, wherein the engineered human T cells are matched with the patient's HLA-B and HLA-C alleles.

[0149] Another aspect of the present invention provides a cell population comprising the engineered human T cells of the present invention.

[0150] In some embodiments, as measured by flow cytometry, the percentage of TRAC, HLA-A and / or TCR protein-negative cells in the cell population is at least 65%, preferably at least 80%, more preferably at least 90%, particularly more preferably at least 95%, or most preferably at least 99%.

[0151] Another aspect of the present invention provides a kit comprising:

[0152] a) An engineered human T cell comprising:

[0153] i. By modifying the gene in the T cell receptor α chain gene, reduce or eliminate the surface layer of the endogenous T cell receptor α chain.

[0154] Express;

[0155] ii. By modifying the HLA-A gene, the surface expression of HLA-A is reduced or eliminated compared to unmodified T cells;

[0156] iii. By modifying the CIITA gene, the surface expression of HLA class II can be reduced or eliminated, and

[0157] iv. Switchable chimeric antigen cell surface receptors, including:

[0158] Tags combined with fields or tags,

[0159] Extracellular hinges and transmembrane domains, and

[0160] Signal transduction structural domain, and

[0161] b) A targeting module comprising a tag-binding domain or tag, and at least one target cell binding domain, or a nucleic acid, vector, or cell encoding the targeting module.

[0162] The tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

[0163] The term "targeting module" refers to a polypeptide or protein having at least two distinct domains, each domain being specific for a target or a set of identical targets, wherein at least one domain is specific for target cells, and one domain is specific for a switchable chimeric antigen receptor (particularly specific for a tag-binding domain or tag). In some embodiments, the targeting module is isolated. The term "isolated" means altered or removed from its natural state.

[0164] Preferably, the targeting module is expressed as a recombinant protein. In some embodiments, the targeting module is chemically synthesized.

[0165] The term "specificity" refers to the ability of an antibody or antibody fragment or protein, peptide or low molecular weight organic ligand to recognize and bind to a binding target (e.g., tumor antigen) protein present in a sample, but not to recognize or bind to other molecules in the sample.

[0166] The term "bonding" refers to non-covalent bonding, particularly ionic bonds, hydrogen bonds, van der Waals forces, and / or hydrophobic interactions.

[0167] In some embodiments, the targeting module is in the form of a monomer, dimer, or polymer, preferably in the form of a monomer.

[0168] In some embodiments, the targeting module is monovalent, bivalent, or multivalent.

[0169] The term "target cell binding domain" is a peptide, protein, or low molecular weight organic ligand that specifically binds to a protein or protein complex (antigen) on the surface of a target cell (preferably a cancer cell, T cell, infected cell, pathogen, or parasite).

[0170] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence.

[0171] The term "low molecular weight organic ligand" refers to an organic molecule with a maximum molecular weight of 10,000 Daltons, preferably 3,000 Daltons, which can specifically bind to proteins or protein complexes (antigens) on the surface of target cells (preferably cancer cells, T cells, infected cells, pathogens, or parasites).

[0172] In some embodiments, the at least one target cell binding domain is an antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD 123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, and CD371; cytokine receptors, preferably interleukin receptors, particularly IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, and IL-13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and their mutants, particularly preferred. ErbB1, ErbB2, ErbB3, ErbB4 or their mutants; members of the tumor necrosis factor receptor superfamily, tight junction proteins, hepatin, hepatin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate-specific antigen, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptors; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin family, etc. FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, NKG2D receptor ligands, epithelial cell glycoprotein family members, disialotyl ganglioside, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus proteins, cancer-testis antigens, fibroblast activating proteins, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans, including mutants and analogs of specified antibodies, antibody fragments, proteins, peptides, or low molecular weight organic ligands.

[0173] The term "mutant" refers to a peptide or protein that has at least 90% sequence identity with a specified antibody, antibody fragment, protein, or peptide, preferably at least 95% sequence identity. Preferably, the mutant binds to the same antigen as the specified antibody, antibody fragment, protein, or peptide.

[0174] The term "analyte" refers to a molecule that has a high degree of structural similarity to a specified antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand, preferably in which at least one atom, group, functional group, or substructure is substituted by another group (e.g., a hydroxyl group). In some embodiments, the analogue of somatostatin (SRIF14) is octreotide or pasireotide. Preferably, the analogue binds to the same antigen as the specified antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand.

[0175] In some embodiments, the specified antibody, antibody fragment, protein, or peptide analogue comprises a selection from those containing modifications such as: D amino acids, pseudopeptide bonds, amino alcohols, non-proteinogenic amino acids, non-natural amino acids, amino acids having modified side chains, and / or cyclic proteins. Preferably, these analogues exhibit increased stability.

[0176] The term "target cell binding domain" also includes soluble T-cell receptors, which consist of α and β or γ and δ chains of the T-cell receptor (TCR), fragments thereof, or mutants thereof. Such TCR-derived binding moieties recognize and bind peptides presented by human leukocyte antigen (HLA) class I and II protein complexes. Examples (but not limited to) TCRs specific to peptides derived from proteins including the EGFR family, survivin, the SRY-like high-mobility group B (SOX) protein family, melanoma-associated antigens (e.g., autoimmune cancer / testis antigen NY-ESO-1, melanoma antigen family member A MAGEA, and the preferentially expressed antigen PRAME in melanoma), and leukemia-associated antigens (e.g., Wilms' tumor gene 1, WT1).

[0177] In some embodiments, the target cell binding domain is a soluble T cell receptor composed of the α and β chains or the γ and δ chains of the T cell receptor (TCR).

[0178] In some embodiments, at least one target cell binding domain is an antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, and CD371, cytokine receptors, CX... CR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and their mutants, members of the tumor necrosis factor receptor superfamily, tight junction proteins, hepatin, hepatin receptor, fucosyltransferase, prostate-specific antigen, embryonic antigen, members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial cell glycoprotein family, disialotetrahexosylganglioside, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus protein, cancer-testis antigen, fibroblast activating protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans.

[0179] In some embodiments, the at least one target cell binding domain is an antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand that binds to CD123.

[0180] In some embodiments, the variable region of the at least one target cell binding domain (preferably a CD123 binding domain) contains a humanized amino acid sequence.

[0181] In some embodiments, the at least one target cell binding domain is an antibody fragment that binds to CD123.

[0182] In some embodiments, the targeting module is bivalent or multivalent and includes at least one CD123 binding domain.

[0183] In some embodiments, different domains of the targeting module are interconnected via linkers. The linkers comprise short sequences, preferably 20 to 30 amino acid residues. In some embodiments, the targeting module comprises flexible peptide sequences selected such that the domains exhibit three-dimensional folding, enabling them to exhibit specificity for effector and target cell binding pairs.

[0184] The preferred connector is one with (G) x S y The linker is a glycine-serine linker with a structure of 1 to 10, preferably 1 to 5. Most preferably, it is a repeating sequence of 2 to 5 sequences of G4S1 (SEQ ID No. 4). Furthermore, the preferred linker can enhance the protease resistance of the antibody derivative.

[0185] In some embodiments, the linker of the tag-binding domain contains 20 to 30 amino acids, preferably 25 amino acids.

[0186] In some embodiments, the connector has a sequence as shown in SEQ ID No. 5 or SEQ ID No. 6.

[0187] In some embodiments, the targeting module comprises one sequence from the sequences shown in SEQ ID No. 19 to SEQ ID No. 32.

[0188] In some embodiments, the targeting module comprises one sequence from the sequences shown in SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 31, or SEQ ID No. 32. In some embodiments, the sequence of the targeting module is as shown in SEQ ID No. 24 or SEQ ID No. 31.

[0189] In some embodiments, the targeting module includes another domain selected from: co-stimulatory ligands, radionuclides, cell-death-inducing compounds, and half-life-extending domains, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, FcRn-binding peptides, or mutants thereof. The term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95%, with the half-life-extending domain. Preferably, the mutant is capable of possessing one or more activities of the specified peptide or protein, particularly extending the half-life like the half-life-extending domain.

[0190] In some embodiments, the targeting module includes another domain selected from: co-stimulatory ligands, radionuclides, cell death-inducing compounds, and half-life-extending domains, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, or FcRn-binding peptides.

[0191] In some embodiments, the length of the targeting module ranges from 20 to 1600 amino acids, preferably from 200 to 800 amino acids.

[0192] In some embodiments, the kit further comprises at least one additional targeting module, or at least one nucleic acid, vector, or cell encoding the additional targeting module.

[0193] The at least one additional targeting module comprises at least one target cell binding domain and a tag binding domain or tag.

[0194] The at least one target cell binding domain is an antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150. CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, and CD371; cytokine receptors, preferably interleukin receptors, particularly IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, and IL-13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and their mutants, particularly ErbB1, ErbB2, ErbB3, ErbB4, or their mutants; tumor necrosis factor receptor superfamily members, tight junction proteins, hepatin, and hepatin receptors, particularly EphA1-10, EphA5, or EphB1-6; fucosyltransferase and prostate-specific antigen, preferably... Selected as PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrin, members of the mucin family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of NKG2D receptor, members of the epithelial cell glycoprotein family, disialotetrahexosylganglioside, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus protein, cancer-testis antigen, fibroblast activating protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans, including mutants and analogs of specified antibodies, antibody fragments, proteins, peptides, or low molecular weight organic ligands.

[0195] The targeting module and the at least one additional targeting module contain different target cell binding domains and the same tag binding domain or tag.

[0196] In some embodiments, at least one additional targeting module includes two target cell binding domains that bind to the following surface antigens, respectively: PSCA and PSMA, ErbB-1 and ErbB-2, PSCA and ErbB-2, PSMA and CEA, IL13Rα2 and ErbB-2, CD38 and CD269, CD19 and CD20, mesothelin and mucin 16, PD-L1 and ErbB-2.

[0197] In some embodiments, the kit contains one to three targeting modules, preferably one CD123-binding targeting module and one or two additional targeting modules.

[0198] In some embodiments, the kit comprises nucleic acids, vectors, or cells encoding a targeting module. The nucleic acids, vectors, and / or cells are isolated.

[0199] In some embodiments, the nucleic acid is cDNA. The term "cDNA" (complementary DNA) refers to a double-stranded DNA synthesized from single-stranded RNA, such as mRNA, through a reaction catalyzed by reverse transcriptase. In some embodiments, cDNA is synthetic. In some embodiments, cDNA is derived from mRNA and therefore, unlike genomic DNA, contains only exons and no introns.

[0200] The vector is preferably a plasmid, artificial chromosome, linearized DNA or RNA, viral particle, or other vector containing an expression cassette, which can be stably integrated into the host cell or host organism genome.

[0201] In some embodiments, the cells are selected from immune cells, preferably immune cells with cytolytic, phagocytic, or immunosuppressive activities, such as T cells, natural killer (NK) cells, and macrophages. In some embodiments, the cells are selected from T cells, including α / β and γ / δ T cells or T cell subsets, such as stem cell memory T cells or central memory T cells, cytotoxic T cells, or NK cells.

[0202] In some embodiments, engineered human T cells and / or targeting modules are presented in the form of pharmaceutical compositions.

[0203] In some embodiments, the kit is used in methods for stimulating chimeric antigen receptor-mediated immune responses in mammals, preferably for treating cancer, infectious diseases, or autoimmune diseases.

[0204] In some embodiments, engineered human T cells and / or targeting modules are used to prepare medicaments for therapeutic and / or diagnostic purposes for cancer, infection, or autoimmune diseases.

[0205] In some embodiments, engineered human T cells and / or targeting modules are used to treat tumors expressing CD123, preferably acute myeloid leukemia.

[0206] Another aspect of the present invention is a kit comprising:

[0207] a) A CRISPR system comprising:

[0208] Nucleic acid targeting a specific region of the endogenous T cell receptor α chain gene.

[0209] Nucleic acid targeting a specific region of the endogenous HLA-A gene.

[0210] Nucleic acids targeting a specific region of the endogenous CIITA gene, and

[0211] b) A nucleic acid or a vector containing a nucleic acid, wherein the nucleic acid-encoded switchable chimeric antigen cell surface receptor comprises:

[0212] Tags combined with fields or tags,

[0213] Extracellular hinges and transmembrane domains, and

[0214] Signal transduction structural domain, and

[0215] c) A targeting module, or a nucleic acid, vector, or cell encoding the targeting module, said targeting module comprising a tag-binding domain or a tag and at least one target cell binding domain.

[0216] The tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

[0217] The term "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats) refers to a bacterial adaptive immune system used to defend against viral invasion. Upon contact with a virus, short segments of viral DNA are integrated into CRISPR loci in the bacterial genome. RNA is transcribed from a portion of the viral sequence contained at the CRISPR locus. This RNA contains a sequence complementary to the viral genome, mediating the targeting of the Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves the viral target, thus silencing it. The CRISPR / Cas system has been used for genome editing in eukaryotic cells. Introducing site-specific single-strand breaks (SSBs) or double-strand breaks (DSBs) can alter the target sequence through, for example, non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0218] In some embodiments, the CRISPR system comprises CRISPR-associated protein 9 mRNA and site-specific single-guide RNA targeting the T-cell receptor α constant region gene (TRAC), human leukocyte antigen A (HLA-A), or CIITA, encapsulated in lipid nanoparticles.

[0219] In some embodiments, the site-specific guide RNA targeting a region of the endogenous T cell receptor α chain gene comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0220] In some embodiments, the site-specific single-stranded guide RNA targeting a region of HLA-A comprises a nucleotide sequence as shown in SEQ ID NO. 2.

[0221] In some embodiments, the site-specific single-stranded guide RNA that causes downregulation of CIITA gene expression comprises a nucleotide sequence as shown in SEQ ID NO.3.

[0222] Table 1. Exemplary Guide RNA

[0223]

[0224]

[0225] Another aspect of the present invention is a method for preparing engineered human T cells expressing switchable chimeric antigen receptors, the method comprising:

[0226] (1) Provide human T cells,

[0227] (2) Introducing a CRISPR system into human T cells by transfection, the CRISPR system containing nucleic acids that target the α chain region of the endogenous T cell receptor in T cells.

[0228] (3) Introducing a CRISPR system into the human T cells via transfection, the CRISPR system containing nucleic acids targeting the HLA-A region of T cells.

[0229] (4) Introducing a CRISPR system into the human T cells via transfection, the CRISPR system containing nucleic acids targeting the CIITA region of T cells.

[0230] This led to the preparation of CRISPR-modified T cells, and

[0231] (5) Introducing a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing a nucleic acid encoding a switchable chimeric antigen cell surface receptor into human T cells via transduction, wherein the switchable chimeric antigen cell surface receptor comprises:

[0232] Tags combined with fields or tags,

[0233] Extracellular hinges and transmembrane domains, and

[0234] Signal transduction structural domain.

[0235] In some embodiments, the switchable chimeric antigen cell surface receptor is a reverse universal chimeric antigen cell surface receptor that includes a tag, an extracellular hinge, a transmembrane domain, and a signal transduction domain.

[0236] In some embodiments, the CRISPR system includes a lipid nucleic acid assembly composition comprising a nuclease or mRNA encoding a nuclease and a site-specific single-stranded guide RNA.

[0237] In some embodiments, the lipid-nucleic acid assembly composition comprises lipid nanoparticles. In some embodiments, the lipid-nucleic acid assembly composition is lipid nanoparticles (LNPs).

[0238] In some embodiments, the lipid nanoparticles are a mixture of cationic lipids and / or ionizable lipids with auxiliary lipids.

[0239] In some embodiments, the lipid nanoparticles are a mixture of ionizable lipids, auxiliary lipids, and PEG-DMG.

[0240] In some embodiments, the endonuclease is CRISPR-associated enzyme 9.

[0241] In some embodiments, the endonuclease is *Streptococcus pyogenes* Cas9, *Neisseria meningitidis* Cas9, *Streptococcus thermophilus* Cas9, or *Staphylococcus aureus* Cas9.

[0242] In this invention, the method includes introducing a CRISPR system via transfection, the CRISPR system containing nucleic acids that can downregulate or eliminate the expression of the TRAC gene in T cells.

[0243] In some embodiments, gene modification of the TRAC gene includes modifying at least one nucleotide in the genomic coordinates chr14:22547524-chr14:22547544. In some embodiments, gene modification of the TRAC gene includes modifying at least 10 or at least 15 consecutive nucleotides in the said genomic coordinates.

[0244] In some embodiments, genetic modification of the TRAC gene involves modifying at least one nucleotide of an exon of the TRAC gene.

[0245] In some embodiments, genetic modification of the TRAC gene involves modifying at least one nucleotide in the genomic coordinates with at least one type of modification, including insertion, deletion, substitution, or deamination.

[0246] In some embodiments, genetic modifications to the TRAC gene include insertions / deletions.

[0247] In some embodiments, the expression of the TRAC gene is reduced or eliminated by binding a gene editing system to a TRAC genomic target sequence, the target sequence comprising at least five consecutive nucleotides in genomic coordinates, preferably ranging from chr14:22547524 to chr14:22547544.

[0248] In some embodiments, the site-specific single-stranded guide RNA in step (2) comprises a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having at least 90%, preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID NO.1.

[0249] In this invention, the method includes introducing a CRISPR system by transfection and downregulation of gene expression, wherein the CRISPR system contains nucleic acids that target a certain region of HLA-A in T cells.

[0250] In some embodiments, the genetic modification of the HLA-A gene includes modifying at least one nucleotide in genomic coordinates selected from chr6:29942854-chr6:29942913 and chr6:29943518-chr6:29943619.

[0251] In some embodiments, the HLA-B genotype of the T cells is homozygous, and / or the HLA-C genotype of the T cells is homozygous. Preferably, both the HLA-B and HLA-C genotypes of the T cells are homozygous.

[0252] In some embodiments, the expression of at least one HLA-A allele selected from HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24 is reduced or eliminated in T cells.

[0253] In some embodiments, the genetic modification of the HLA-A gene includes modifying at least one nucleotide in the genomic coordinates chr6:29942864-chr6:29942903 (preferably chr6:29942876-chr6:29942897).

[0254] In some embodiments, the genetic modification of the HLA-A gene includes modifying at least one nucleotide in the genomic coordinates chr6:29943528-chr6:29943609 (preferably chr6:29943528-chr6:29943550).

[0255] In some embodiments, gene modification of the HLA-A gene includes modifying at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides in the genomic coordinates, preferably modifying at least 10, 15, 16, 17, 18, 19, or 20 consecutive nucleotides in the genomic coordinates.

[0256] In some embodiments, the genetic modification of the HLA-A gene includes at least one C to T substitution or at least one A to G substitution in the genomic coordinates.

[0257] In some embodiments, the genetic modification of the HLA-A gene includes insertions / deletions.

[0258] In some embodiments, a gene editing system is used to bind to an HLA-A genomic target sequence to reduce or eliminate HLA-A expression. The HLA-A genomic target sequence comprises at least five consecutive nucleotides in genomic coordinates selected from the ranges of: chr6:29942864-29942884, chr6:29942868-29942888, chr6:29942876-29942896, chr6:29942877-29942897, chr6:29942 883-29942903, chr6:29943126-29943146, chr6:29943528-29943548, chr6:29943529-29943549, chr6:29943530-29943550, chr6:29943537-29943557, chr6:29943549-29943569, chr6:29943589-29943609 and chr6:29944026-29944046.

[0259] In certain embodiments, the HLA-B allele is selected from any one of the following: HLA-B*07:02, HLA-B*08:01, HLA-B*44:02, HLA-B*35:01, HLA-B*40:01, HLA-B*57:01, HLA-B*14:02, HLA-B*15:01, HLA-B*13:02, HLA-B*44:03, HLA-B*38:01, HLA-B*18:01, HLA-B*44:03, HLA-B*51:01, HLA-B*49:01, HLA-B*15:01, HLA-B*18:01, HLA-B*27:05, HLA-B*35:03, HLA-B*18:01, HLA-B*52:01, HLA-B*51:01, HLA-B*37:01, HLA-B*53:01, HLA-B*55:01, HLA-B*44:02, HLA-B*44:03, HLA-B*35:02, HLA-B*15:01, and HLA-B*40:02.

[0260] In certain embodiments, the HLA-C allele is selected from any one of the following: HLA-C*07:02, HLA-C*07:01, HLA-C*05:01, HLA-C*04:01, HLA-C*03:04, HLA-C*06:02, HLA-C*08:02, HLA-C*03:03, HLA-C*06:02, HLA-C*16:01, HLA-C*12:03, HLA-C*07:01, HLA-C*04:01, HLA-C*15:02, HLA-C*07:01, HLA-C*03:04, HLA-C*12:03, HLA-C*02:02, HLA-C*04:01, HLA-C*05:01, HLA-C*12:02, HLA-C*14:02, HLA-C*06:02, HLA-C*04:01, HLA-C*03:03, HLA-C*07:04, HLA-C*07:01, HLA-C*04:01, HLA-C*04:01, and HLA-C*02:02.

[0261] In certain embodiments, the HLA-B allele is selected from any one of the following: HLA-B*07:02, HLA-B*08:01, HLA-B*44:02, HLA-B*35:01, HLA-B*40:01, HLA-B*57:01, HLA-B*14:02, HLA-B*15:01, HLA-B*13:02, HLA-B*44:03, HLA-B*38:01, HLA-B*18:01, HLA-B*44:03, HLA-B*51:01, HLA-B*49:01, HLA-B*15:01, HLA-B*18:01, HLA-B*27:05, HLA-B*35:03, HLA-B*18:01, HLA-B*52:01, HLA-B*51:01, HLA-B*37:01, HLA-B*53:01, HLA-B*55:01, HLA-B*44:02, HLA-B*44:03, HLA-B*35:02, HLA-B*15:01, and HLA-B*40:02, and the HLA-C allele is selected from any one of the following: HLA-C*07:02, HLA-C*07:01, HLA-C*05:01, HLA-C*04:01, HLA-C*03:04, HLA-C*06:02, HLA-C*08:02, HLA-C*03:03, HLA-C*06:02, HLA-C*16:01, HLA-C*12:03, HLA-C*07:01, HLA-C*04:01, HLA-C*15:02, HLA-C*07:01, HLA-C*03:04, HLA-C*12:03, HLA-C*02:02, HLA-C*04:01, HLA-C*05:01, HLA-C*12:02, HLA-C*14:02, HLA-C*06:02, HLA-C*04:01, HLA-C*03:03, HLA-C*07:04, HLA-C*07:01, HLA-C*04:01, HLA-C*04:01, and HLA-C*02:02.

[0262] In some embodiments, the HLA-B and HLA-C alleles are selected from any of the following groups: HLA-B*07:02 and HLA-C*07:02, HLA-B*08:01 and HLA-C*07:01, HLA-B*44:02 and HLA-C*05:01, HLA-B*35:01 and HLA-C*04:01, HLA-B*40:01 and HLA-C*03:04, HLA-B*57:01 and HLA-C*06:02, HLA-B*14:02 and H LA-C*08:02, HLA-B*15:01 and HLA-C*03:03, HLA-B*13:02 and HLA-C*06:02, HLA-B*44:03 and HLA-C*16:01, HLA-B*38:01 and HLA-C*12:03, HLA-B*18:01 and HLA-C*07:01, HLA-B*44:03 and HLA-C*04:01, HLA-B*51:01 and HLA-C*15:02, HLA-B*49: 01 and HLA-C*07:01, HLA-B*15:01 and HLA-C*03:04, HLA-B*18:01 and HLA-C*12:03, HLA-B*27:05 and HLA-C*02:02, HLA-B*3 5:03 and HLA-C*04:01, HLA-B*18:01 and HLA-C*05:01, HLA-B*52:01 and HLA-C*12:02, HLA-B*51:01 and HLA-C*14:02, HLA-B *37:01 and HLA-C*06:02, HLA-B*53:01 and HLA-C*04:01, HLA-B*55:01 and HLA-C*03:03, HLA-B*44:02 and HLA-C*07:04, HLA -B*44:03 and HLA-C*07:01, HLA-B*35:02 and HLA-C*04:01, HLA-B*15:01 and HLA-C*04:01, and HLA-B*40:02 and HLA-C*02:02.

[0263] In some embodiments, the T cells further comprise HLA-B surface expression reduced or eliminated relative to unmodified T cells via gene modification in the HLA-B gene, and / or HLA-C surface expression reduced or eliminated relative to unmodified T cells via gene modification in the HLA-C gene.

[0264] In some embodiments, the T cells also include HLA-B surface expression reduced or eliminated relative to unmodified T cells through genetic modifications in the HLA-B gene, and the HLA-C genotype of the T cells is homozygous.

[0265] In an alternative embodiment, the T cells also include HLA-C surface expression reduced or eliminated relative to unmodified T cells through genetic modification in the HLA-C gene, and the HLA-B genotype of the T cells is homozygous.

[0266] In some embodiments, the T cells also include gene modifications in the HLA-B and HLA-C genes that reduce or eliminate the surface expression of HLA-B and HLA-C compared to unmodified T cells.

[0267] In some embodiments, the site-specific single-stranded guide RNA in step (3) comprises a nucleotide sequence as shown in SEQ ID NO.2, or a sequence having at least 90%, preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID NO.2.

[0268] In some embodiments, the HLA-A guide RNA contains at least one modification selected from the following types: 2'-O-methyl (2'-O-Me) modified nucleotides, phosphate thioester (PS) bonds between nucleotides, 2'-fluorine (2'-F) modified nucleotides, modifications of one or more nucleotides of the first five nucleotides at the 5' end of the guide RNA, modifications of one or more nucleotides of the last five nucleotides at the 3' end of the guide RNA, PS bonds between the first four nucleotides of the guide RNA, PS bonds between the last four nucleotides of the guide RNA, 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the guide RNA, 2'-O-Me modified nucleotides at the last three nucleotides at the 3' end of the guide RNA, or combinations of the above modifications.

[0269] In some embodiments, the method further includes matching engineered human T cells with HLA-B and HLA-C alleles to a patient.

[0270] In alternative embodiments, the method further includes introducing a CRISPR system containing nucleic acids targeting the HLA-B region of T cells into human T cells via transfection and downregulation of gene expression, and / or introducing a CRISPR system containing nucleic acids targeting the HLA-C region of T cells into human T cells.

[0271] In this invention, the method includes introducing a CRISPR system via transfection, the CRISPR system containing nucleic acids that target the CIITA region in T cells.

[0272] In some embodiments, genetic modification of the CIITA gene includes modifying at least one nucleotide at a splice site in genomic coordinates chr16:10902171-chr16:10923242.

[0273] In some embodiments, genetic modification of the CIITA gene includes modification of at least one nucleotide at the splice acceptor site, preferably wherein the nucleotide is A, G, or T.

[0274] In some embodiments, genetic modifications to the CIITA gene include modifications to the splice site boundary nucleotides.

[0275] In some embodiments, genetic modification of the CIITA gene involves modifying at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides at genomic coordinates chr16:10902171-chr16:10923242.

[0276] In some embodiments, genetic modifications to the CIITA gene include at least one C to T substitution or at least one A to G substitution in genomic coordinates chr16:10902171-chr16:10923242.

[0277] In some embodiments, genetic modifications to the CIITA gene include insertions / deletions, C-to-T substitutions, or A-to-G substitutions selected from the following genomic coordinates: chr16:10895410-10895430, chr16:10898649-10898669, chr16:10898658-10898678, chr16:10902171-10902191, chr16:10902173-10902193, chr16:10902174-10902194, chr16:10902179-10902199, chr16:10902183-10902203 ... r16:10902184-10902204, chr16:10902644-10902664, chr16:10902779- 10902799, chr16:10902788-10902808, chr16:10902789-10902809, chr16 :10902790-10902810, chr16:10902795-10902815, chr16:10902799-1090 2819、chr16:10903708-10903728、chr16:10903713-10903733、chr16:109 03718-10903738、chr16:10903721-10903741、chr16:10903723-1090374 3. chr16:10903724-10903744, chr16:10903873-10903893, chr16:109038 78-10903898, chr16:10903905-10903925, chr16:10903906-10903926, ch r16:10904736-10904756, chr16:10904790-10904810, chr16:10904811-1 0904831, chr16:10906481-10906501, chr16:10906485-10906505, chr16: 10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906 512、chr16:10908127-10908147、chr16:10908130-10908150、chr16:1090 8131-10908151, chr16:10908132-10908152, chr16:10908137-10908157,chr16:10908138-10908158, chr16:10908139-10908159, chr16:10909006-10909026, chr16:10909007-10909027, chr16:10909018-109 09038, chr16:10909021-10909041, chr16:10909022-10909042, chr16:10909172-10909192, chr16:10910165-10910185, chr16:109101 76-10910196、chr16:10910186-10910206、chr16:10915547-10915567、chr16:10915551-10915571、chr16:10915552-10915572、chr16: 10915567-10915587、chr16:10916348-10916368、chr16:10916359-10916379、chr16:10916362-10916382、chr16:10916449-10916469、 chr16:10916450-10916470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:10918423-10918443, chr16:10918504-109 18524, chr16:10918511-10918531, chr16:10918512-10918532, chr16:10918539-10918559, chr16:10922153-10922173, chr16:109224 78-10922498, chr16:10922487-10922507, chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241, and chr16:10923222-10923242.

[0278] In some embodiments, the genetic modification of the CIITA gene comprises at least five consecutive nucleotides selected from the following genomic coordinates: chr16:10906485-10906505, chr16:10906486-10906506, chr16:10906487-10906507, chr16:10906492-10906512, chr16:10908127-10908147, chr16:10908130-10908150, chr16:10908131-10908151, chr16:10908132-10908152, chr16:1 0908137-10908157, chr16:10908138-10908158, chr16:10908139-10908 159. chr16:10909006-10909026, chr16:10909007-10909027, chr16:1090 9018-10909038, chr16:10909021-10909041, chr16:10909022-10909042 , chr16:10909172-10909192, chr16:10910165-10910185, chr16:1091017 6-10910196, chr16:10910186-10910206, chr16:10915547-10915567, ch r16:10915551-10915571, chr16:10915552-10915572, chr16:10915567- 10915587、chr16:10916348-10916368、chr16:10916359-10916379、chr1 6:10916362-10916382, chr16:10916449-10916469, chr16:10916450-109 16470, chr16:10916455-10916475, chr16:10916456-10916476, chr16:1 0918423-109l8443、chr16:10918504-10918524、chr16:10918511-109185 31. chr16:10918512-10918532, chr16:10918539-10918559, chr16:1092 2153-10922173、chr16:10922478-10922498、chr16:10922487-10922507、chr16:10922499-10922519, chr16:10923205-10923225, chr16:10923214-10923234, chr16:10923218-10923238, chr16:10923219-10923239, chr16:10923220-10923240, chr16:10923221-10923241 and chr16: 10923222-10923242, preferably chr16:10908132-10908152, chr16:10908131-10908151, chr16:10916456-10916476, chr16:10918504-10918524, chr16:10909022-10909042, chr16:10918512-10918532, chr16:1091 8511-10918531, chr16:10895742-10895762, chr16:10916362-10916382, chr16:10916455-10916475, c hr16:10909172-10909192, chr16:10906492-10906512, chr16:10909006-10909026, chr16:10922478-10 922498, chr16:10895747-10895767, chr16:10916348-10916368, chr16:10910186-10910206, chr16:10906481-10906501, chr16:10909007-10909027, chr16:10895410-10895430 and chr16:10908130-10908150.

[0279] In some embodiments, genetic modification of the CIITA gene involves modifying at least 10 or at least 15 consecutive nucleotides in the genomic coordinates.

[0280] In some embodiments, the site-specific single-stranded guide RNA in step (4) is a CIITA genomic target sequence containing at least one nucleotide of a splice acceptor site or a splice donor site.

[0281] In some embodiments, the nucleotide is a splice site boundary nucleotide located at a splice acceptor site or a splice donor site.

[0282] In some embodiments, the site-specific single-stranded guide RNA in step (4) comprises a guide sequence that guides the RNA-directed DNA binder to cut the CIITA genomic target sequence at a distance of 4 or fewer, 3 or fewer, 2 or fewer, or 1 nucleotide from the splice site boundary nucleotide.

[0283] In some embodiments, the site-specific single-stranded guide RNA in step (4) comprises a nucleotide sequence as shown in SEQ ID NO.3, or a sequence having at least 90%, preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID NO.3.

[0284] In some embodiments, the CIITA guide RNA contains at least one modification selected from the following types: 2'-O-methyl (2'-O-Me) modified nucleotides, phosphate thioester (PS) bonds between nucleotides, 2'-fluorine (2'-F) modified nucleotides, modifications of one or more nucleotides of the first five nucleotides at the 5' end of the guide RNA, modifications of one or more nucleotides of the last five nucleotides at the 3' end of the guide RNA, PS bonds between the first four nucleotides of the guide RNA, PS bonds between the last four nucleotides of the guide RNA, 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the guide RNA, 2'-O-Me modified nucleotides at the last three nucleotides at the 3' end of the guide RNA, or combinations thereof.

[0285] In some embodiments, genetic modifications to the CIITA gene include insertions / deletions.

[0286] In some embodiments, the introduction of a nucleic acid encoding a switchable chimeric antigen cell surface receptor, or a vector containing said nucleic acid, according to step (5), is achieved by adding a non-viral or viral vector (including, but not limited to, lentiviral or adenoviral vectors). The introduction of the nucleic acid encoding a switchable chimeric antigen cell surface receptor, or a vector containing said nucleic acid, according to step (5) can occur at random sites or in a site-specific manner, including (but not limited to) homology-directed repair (HDR) at CRISPR-edited loci.

[0287] In some embodiments, according to step (5), a nucleic acid encoding a switchable chimeric antigen cell surface receptor or a vector containing said nucleic acid is introduced by adding self-inactivating retroviral vector particles.

[0288] Another aspect of the invention provides a method for stimulating a chimeric antigen receptor-mediated immune response in mammals (preferably humans) suffering from cancer, infectious diseases, or autoimmune diseases by administering the engineered human T cells of the invention, along with a targeting module, pharmaceutical composition, or kit, to a subject in need.

[0289] Another aspect of the invention relates to the use of engineered human T cells and targeting modules, pharmaceutical compositions or kits of the invention for stimulating chimeric antigen receptor-mediated immune responses in mammals (preferably humans) suffering from cancer, infectious diseases or autoimmune diseases.

[0290] To treat the above-mentioned diseases, a subject is administered the sterile pharmaceutical composition or sterile kit of the present invention, which comprises a pharmacologically effective amount of the engineered human T cells and targeting module of the present invention.

[0291] In some embodiments, a method for stimulating a chimeric antigen receptor-mediated immune response in mammals (preferably a method for treating cancer, infectious diseases, or autoimmune diseases) includes the following steps:

[0292] a) Administering an effective amount of the targeting module to mammals, and

[0293] b) Administering an effective amount of the engineered human T cells of the present invention to a mammal.

[0294] The tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor, or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

[0295] Specifically, the targeting module is administered to mammals before, simultaneously with, or after the administration of the vector or cells.

[0296] In some embodiments, the method for stimulating a chimeric antigen receptor-mediated immune response in mammals further includes: identifying the HLA-B and HLA-C alleles of a subject (patient), and matching the HLA-B and HLA-C alleles of engineered human T cells with those of the patient (Furst et al., 2019).

[0297] Matching the HLA-B and HLA-C alleles of engineered human T cells with the patient is a crucial step because (HLA) mismatch is a significant risk factor for antibody-mediated and immune cell-mediated rejection responses, as well as transplant failure. Matched alleles refer to identical alleles as understood in related technologies.

[0298] In some embodiments, the patient carries the same HLA-B and HLA-C alleles as engineered human T cells.

[0299] In an alternative embodiment, the patient carries one or more HLA-B and HLA-C alleles from engineered human T cells.

[0300] In some embodiments, a method for stimulating a chimeric antigen receptor-mediated immune response in mammals (preferably for treating cancer, infectious diseases, or autoimmune diseases) includes the following steps:

[0301] a) Administering an effective amount of a targeting module to a mammal, said targeting module comprising at least one target cell binding domain and a tag binding domain or tag.

[0302] The at least one target cell binding domain is an antibody, antibody fragment, protein, peptide, or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150. CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366, and CD371; cytokine receptors, preferably interleukin receptors, particularly IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, and IL-13Rα1; CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and their mutants, particularly ErbB1, ErbB2, ErbB3, ErbB4, or their mutants; tumor necrosis factor receptor superfamily members, tight junction proteins, hepatin, and hepatin receptors, particularly EphA1-10, EphA5, or EphB1-6; fucosyltransferase and prostate-specific antigen, preferably... Selected as PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of NKG2D receptor, members of the epithelial cell glycoprotein family, disialotetrahexosylganglioside, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus protein, cancer-testis antigen, fibroblast activating protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans, including mutants and analogs of specified antibodies, antibody fragments, proteins, peptides, or low molecular weight organic ligands, and

[0303] b) Administer an effective amount of engineered human T cells to mammals, and

[0304] c) Administering an effective amount of another targeting module to mammals.

[0305] The targeting module and at least one additional targeting module contain different target cell binding domains, as well as the same tag binding domain or tag, wherein the method is performed in the order of steps a), b), and c).

[0306] In some embodiments, the targeting module is administered alone before the administration of engineered human T cells (preferably 1 hour to 2 days, more preferably 4 to 24 hours), and continuously after the administration of engineered human T cells (preferably within 3 to 30 days). Additionally, an extra dose of the targeting module may be administered after the resting period to reactivate effector cells carrying switchable chimeric antigen receptors.

[0307] This invention is not limited to the embodiments shown, but also includes all embodiments that have equivalent effects within the scope of this invention. Furthermore, this invention is not limited to the specifically described combination of features, but can also be defined by any other combination of specific features of all individual features disclosed in its entirety, provided that the individual features are not mutually exclusive, or that no specific combination of individual features is explicitly excluded.

[0308] The present invention will be explained in more detail below through specific embodiments. These specific embodiments refer to engineered human T cells and their preparation methods, and are intended to describe the invention rather than limit it. Attached Figure Description

[0309] The invention will now be further explained with reference to the following figures and examples.

[0310] Figure 1 A schematic diagram of a switchable chimeric antigen receptor (CAR) with three domains is shown, wherein the first domain is a tag-binding domain or tag (e.g., scFv), the second domain is an extracellular hinge and transmembrane domain, and the third domain is a signal transduction domain. In addition, the extracellular portion of the receptor may also contain an optional fourth domain, namely a short peptide linker.

[0311] Figure 2 This diagram illustrates the synergistic mechanism of engineered T cells and an antigen-specific targeting module (TM) in forming an active drug according to the present invention. Allogeneic T cells expressing RevCAR (Allo-RevCAR-T) carry RevCAR epitopes (RCEs or tags) on their cell surface, preferably short-chain, non-immunogenic peptide motifs derived from human nuclear La / SSB autoantigens. Since ligands binding to RCEs or tags are absent in the human body, Allo-RevCAR-T remains in an inactive mode (left). The antigen specificity of Allo-RevCAR-T is provided via a soluble targeting module (TM) that is specific to the target antigen. The Allo-RevCAR targeting module (TM) consists of a target-binding domain and a tag-binding domain specific to the RCE or tag. The targeting module (TM) activates RevCAR-T effector function by cross-linking RevCAR-T with tumor cells expressing the target antigen, subsequently killing the tumor cells (right).

[0312] Figure 3The transduction efficiency and TRAC, CIITA, and HLA-A knockout frequencies of allo-switchable CAR-T cells are shown. In the figure, A represents the switchable CAR expression frequency, B represents the TCR knockout frequency, C represents the MHC class II knockout frequency, and D represents the HLA-A knockout frequency. The experimental conditions were as follows: (1) LNP-mediated editing of the TRAC-CIITA-HLA-A sequence combined with retroviral vector (RV)-mediated gene transfer; (2) LNP-mediated editing of the TRAC-HLA-A-CIITA sequence combined with RV-mediated gene transfer; and (3) RV-mediated gene transfer only, and (4) LNP-mediated editing of the TRAC locus only, as controls. The editing order could be different, and each point represented a separate donor.

[0313] Figure 4 The protein expression of Ki-67 (a proliferation marker), PD-1 (a depletion marker), LAG-3 (a depletion marker), TIM-3 (a depletion marker), IFN-γ (a depletion marker), and other phenotypic markers in human primary T cells transduced to express switchable CARs (UniCAR or RevCAR) is shown. Statistical analysis was performed using unpaired t-tests, and p-values ​​below 0.05 are shown above each figure.

[0314] Figure 5 This study presents a comparison of the differentiation phenotypes of primary human T cells transduced to express switchable CARs (UniCAR = UC02: n = 10 or RevCAR = RC01: n = 9) after generation. T cells were stained against cell surface markers CD4, CD45RO, CD197, and CD28. Cells were gated according to GFP+ and CD4+ (A, C, E, G, I) or CD4- (B, D, F, H, J), and the percentage of effector / memory cells was determined based on the expression of CD45RO, CD197, and CD28. Statistical analysis was performed using unpaired t-tests; p-values ​​are shown above each figure.

[0315] Figure 6 The images show the cell lysis of CD123-expressing AML cell lines MV4-11 (A, B) or OCI-AML3 (C, D) after 48 hours of treatment with different concentrations of the anti-CD123 targeting module (R-TM123), as measured by flow cytometry. The cells were in the presence of human primary T cells with gene knockout at a switchable CAR (Auto RevCAR-T, A, C) or a CRISPR / Cas-mediated TRAC locus (Allo RevCAR-T, B, D). Dashed lines indicate non-specific lysis in the absence of R-TM123.

[0316] Figure 7This study showcases RevCAR-T products manufactured using a clinical-scale, GMP-compliant process. These products exhibit selective enrichment of CD28 and CC chemokine receptor type 7 (CCR7, CD197) double-positive T cells, which are classified as central memory T cells (Tcm) according to the human T cell subset classification proposed by Mahnke et al. (Mahnke et al., 2013). Therefore, the prepared and frozen final drug sample was thawed and stained with surface markers CD4, CD28, CD45RA, CD45RO, and CD197 (CCR7). Based on the human T cell subset classification proposed by Mahnke et al. (Mahnke et al., 2013), Tcm represents central memory T cells (CD28+CD197+), Ttm represents transitional memory T cells (CD28+CD197-), and Tem represents effector memory T cells (CD28-CD197-).

[0317] Figure 8 The surface expression of CD25 on Allo-RevCAR-T cells was shown under the activation mediated by R-TM123.

[0318] Figure 9 The specific lysis of the AML cell line MOLM-13 by R-TM123-redirected Allo-RevCAR-T was shown.

[0319] Figure 10 The release of cytokines and effector molecules from R-TM123-redirected Allo-RevCAR-T against the AML cell line MOLM-13 is shown.

[0320] Figure 11 The cytotoxic response of R-TM123-redirected Allo-RevCAR-T against primary leukemia cells from AML patients is shown.

[0321] Figure 12 The in vivo pharmacokinetics of R-TM123 in NSG mice were demonstrated.

[0322] Figure 13 The study demonstrated the leukemia elimination effect of R-TM123-redirected Allo-RevCAR-T against extramedullary AML.

[0323] Figure 14 The proportion of RevCAR-positive T cells in Allo-RevCAR-T is shown compared to the unedited RevCAR-T (Auto-RevCAR-T) product. Detailed Implementation

[0324] Gene editing

[0325] Gene editing is achieved by transfecting the target gene-specific sgRNA along with Cas9 mRNA or protein into the cell to be edited. Any transfection method is applicable, including electroporation and lipid transfection or lipid-mediated delivery.

[0326] Guiding choice

[0327] The CRISPR / Cas9 sgRNA target sequence is the 20 nucleotides adjacent to the 5' end of the NGG prespacer motif (PAM). sgRNAs were selected based on genomic loci to target cleavage sites within the coding sequences of TRAC (ENSG00000277734), CIITA (ENSG00000179583), and HLA-A (ENSG00000277734). This excluded sgRNAs that would cleave within introns or the 5' or 3' untranslated regions (UTRs). It also excluded sgRNAs overlapping with high-magnitude allele frequency (MAF) single nucleotide polymorphisms (SNPs) found in the GenomAD database (Lek et al., 2016). Knockout of T-cell receptor α constant region gene (TRAC), MHC class II transactivator (CIITA), and human leukocyte antigen A (HLA-A) was used to screen for a batch of guide RNAs with high computer prediction ranking. These guide RNAs were then screened in primary T cells, and targeted next-generation sequencing (NGS) of polymerase chain reaction (PCR) amplicon sequencing was used for on-target insertion / deletion quantification analysis. The edit percentage (e.g., "edit efficiency," "edit percentage," or "insertion / deletion frequency") was defined as the ratio of the total number of sequencing reads with insertions / deletions or substitutions to the total number of sequencing reads, including wild-type. Nucleotides within ten base pairs of the expected cleavage site were evaluated to determine the presence of insertions, deletions, and mismatches with the reference sequence. Identification results of insertion / deletion quantification of amplicon sequencing using control guides showed a detection limit as low as 0.5%. LNPs containing the most effective guides for each target were tested on primary T cells, and gene knockout efficiency was quantified by NGS and flow cytometry to confirm the loss of protein expression. Figure 3 ).

[0328] Switchable CAR

[0329] To genetically engineer a switchable CAR for expression, a multinucleotide vector encoding the switchable CAR and all the necessary elements to ensure its expression in genetically engineered immune cells must be transferred into the immune cells. Specifically, the switchable CAR contains IL-2LP (modified human IL-2 leader peptide), RCE (RevCAR epitope, also a tag), G4S1 (glycine-serine linker), ECD (extracellular domain), TMD (transmembrane domain), and ICD (intracellular domain).

[0330] Vector transfer can be carried out through electroporation or nucleic acid transfection, or by using viral vector systems such as adenovirus, adeno-associated virus, retrovirus, foam virus, or lentivirus for gene transfer.

[0331] By constructing a lentiviral vector encoding a selected switchable CAR, lentiviral gene transfer was applied to the stable expression of a switchable chimeric antigen receptor CAR in immune cells. The lentiviral vector used was pLVX-EF1αUniCAR 28 / ζ (Clontech, Takara Bio Group), in which the lentiviral portion of the vector was derived from human immunodeficiency virus (HIV), and the MSC / IRES / ZxGreenI portion was replaced with a switchable CAR construct.

[0332] Lentiviral particles were prepared by transiently transfecting human embryonic kidney (HEK) 293T (ACC 635) cells with a lentiviral vector plasmid encoding a switchable CAR, followed by co-transfection with a plasmid (psPAX2) encoding population-specific antigen (gag) and polymerase (pol) and a plasmid encoding the envelope (pMD2.G). After transfection, the packaging plasmid expressed the HIV-1 Gag and Pol proteins. Plasmid MD2.G encodes the vesicular stomatitis virus glycoprotein (VSV-G). The VSV-G protein was used in the lentiviral vector to transduce various mammalian cells. Various envelopes from different viral species could be used for this purpose. The lentiviral vector could be successfully pseudotyped with the envelope glycoprotein (Env) of bitropic murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), a modified envelope of the original foamy virus (PFV), or a chimeric envelope glycoprotein variant derived from gibberish leukemia virus (GaLV) and MLV.

[0333] 24 to 96 hours post-transfection, the supernatant of transfected HEK293T cells was harvested, and viral particles were concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMCs) or isolated T cells were activated using monoclonal antibodies specific to the CD3 complex (e.g., clones OKT3 or UCHT1). These monoclonal antibodies were added in solution or coated onto plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices. Stimulation of the co-stimulatory pathway using monoclonal antibodies or ligands targeting CD27, CD28, CD134, or CD137 (alone or in combination, coated onto plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices), along with the addition of exogenous recombinant cytokines such as interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21, further enhanced the activation of PBMCs or isolated T cells. 24 to 96 hours after initial administration of a single or multiple dose of an activating CD3-specific antibody and / or co-stimulatory receptor CD27, CD28, CD134, or CD137-specific antibody and / or recombinant cytokines, concentrated or unconcentrated viral particles are added to PBMCs or T cell cultures. T cell electroporation, transduction, and expansion can be performed manually in an open cell culture system or in a partially or fully automated closed system.

[0334] Starting on day 3 after the last addition of viral supernatant, the surface expression of the switchable chimeric antigen receptor (CAR) was stained with a tagged molecule or with a monoclonal antibody targeting the fourth domain of the switchable CAR, followed by flow cytometry to detect stable T cell transduction. Switchable CAR-transduced T cells can be expanded in vitro by providing recombinant cytokines and activating an anti-CD3 monoclonal antibody.

[0335] If the switchable CAR contains an optional fourth domain, i.e., a peptide sequence that forms a linear epitope of a monoclonal antibody, then the specific in vitro expansion of genetically modified immune cells expressing the switchable CAR can be achieved by coating a monoclonal antibody or antibody fragment bound to the fourth switchable CAR domain onto the surface of a culture dish, various beads, or a biodegradable polymer matrix, and then adding it to a cell culture at a specified ratio. The binding of the surface-coated monoclonal antibody to the switchable CAR peptide domain induces cross-linking of the switchable CAR expressed on the cell surface and forms an immune synapse, thereby activating a signaling pathway specifically triggered by the signaling domain of the switchable CAR. The activated signaling pathway promotes the proliferation of immune cells carrying the switchable CAR and makes them persistently resistant to activation-induced cell death, thus enriching genetically modified immune cells with the switchable CAR in a mixed cell population.

[0336] An optional fourth domain, a peptide sequence that forms a linear epitope of a monoclonal antibody, can be further used to enrich and purify immune cells expressing a switchable CAR from a mixed cell population. Enrichment and purification are accomplished by binding the switchable CAR fourth domain to a monoclonal antibody or an antibody fragment thereof to label cells expressing the switchable CAR for cell sorting, or by transiently attaching switchable CAR-expressing immune cells to small particles for cell separation. On one hand, immune cells transplanted with switchable CARs are incubated with a monoclonal antibody that recognizes the fourth domain. Then, magnetic beads are added, these beads being coupled with species- and isotype-specific heavy and light chains of the monoclonal antibody that binds to the optional fourth domain. Thus, immune cells expressing the switchable CAR are attached to the magnetic beads and captured in a magnetic field, separating them from other immune cells.

[0337] Preparation of engineered human T cells

[0338] T cells were isolated from fresh leukocytes from healthy human donors who were HLA-B / C genotyped. The purity (CD3+ frequency) and abundance (viability, live cell count) of the selected T cells were characterized. Gene knockout of the T cell receptor α chain (TRAC), HLA-A, and major histocompatibility complex class II transactivator (CIITA) was achieved using lipid nanoparticles (LNPs) containing Cas9 mRNA and single-stranded guide RNA (sgRNA) targeting HLA-A, CIITA, or TRAC. These LNPs consisted of a mixture of lipids (ionizable lipids, helper lipids, cholesterol, and PEGylated lipids) and abundant Cas9-encoding mRNA, along with single-stranded guide RNA targeting the target genes. Sequential addition of LNPs to the culture medium significantly reduced the risk of translocation. The T cell culture medium was genetically modified to express RevCAR by adding self-inactivating retroviral vector particles. After genetic engineering, the culture medium was expanded for 5–6 days before harvesting. TCRα / β+ cells could be eliminated.

[0339] Characterization of engineered human T cells in this invention

[0340] Editing efficiency and RevCAR or UniCAR transduction efficiency were assessed by flow cytometry. Figure 3 The function of the engineered human T cells described in this invention was evaluated, including the protein expression of proliferation markers, exhaustion markers, and other phenotypic markers, as well as the phenotype of primary human T cells after their generation.

[0341] Figure 4This study compares the protein expression of proliferation marker Ki-67, exhaustion markers PD-1, LAG-3, TIM-3, IFN-γ, and other phenotypic markers in primary human T cells transduced to express switchable CARs (UniCAR or RevCAR) (UniCAR or RevCAR). Primary human T cells from four healthy donors were transduced to express switchable CARs (UniCAR = UC02 or RevCAR = RC01) via lentiviral vector-mediated gene transfer. After harvest, the T cells were stained for proliferation marker Ki-67, exhaustion markers PD-1, LAG-3, TIM-3, IFN-γ, and other phenotypic markers. Because the CAR cDNA is followed by a 2pA site and green fluorescent protein (GFP), GFP expression in CD3+ cells was used as a surrogate indicator for CAR positivity. RC01-transduced T cells showed significantly reduced expression of exhaustion markers PD-1 and LAG-3, and a numerical decrease in intracellular IFN-γ expression.

[0342] Figure 5 This study presents a comparison of the differentiation phenotypes of primary human T cells transduced to express switchable CARs (UniCAR or RevCAR) after preparation. Primary human T cells from healthy donors were transduced to express switchable CARs (UniCAR02 = UC02 or RevCAR01 = RC01) via lentiviral vector-mediated gene transfer. Cell surface markers CD4, CD45RO, CD197, and CD28 were stained after harvest on day 14. Gating selection was performed on cells based on GFP+ and CD4+ (A, C, E, G, I) or CD4- (B, D, F, H, J), and the percentage of effector / memory cells was determined based on the expression of CD45RO, CD197, and CD28. RC01-transduced T cells showed an enrichment of undifferentiated memory cells (SCM: stem cell memory cells, CM: central memory cells) and a decreased proportion of highly differentiated cells (TM: transitional memory cells, EM: effector memory cells, LE: late effector cells). Therefore, RevCAR-T cells exhibited a low-differentiation phenotype after preparation.

[0343] Design of the targeting module for the reagent kit of the present invention

[0344] The targeting module R-TM123 is a soluble recombinant fusion protein containing two antibody-derived binding domains. One domain selectively binds to the target antigen CD123, and the other domain recognizes the RCE or tag (epitope E5B9 from the human La protein) presented on RevCAR-expressing cells. Therefore, R-TM123 acts as a bridging module between Allo-RevCAR-T cells and CD123-expressing target cancer cells. Figure 2 The targeting module also includes an 8x-histidine tag at its C-terminus for detection and purification.

[0345] Cytotoxicity assay

[0346] The efficacy of the anti-CD123 targeting module™ in inducing tumor cell elimination via switchable CAR-T cells was tested using flow cytometry-based cytotoxicity assays with AML cell lines MV4-11 or OCI-AML3. Figure 6 Primary human T cells from the same healthy donor were transduced via lentiviral vector-mediated gene transfer to express switchable CARs (AutoRevCAR-T) or additionally edited via CRISPR / Cas-mediated TRAC locus knockout (AlloRevCAR-T). The potency of the targeting module TM was assessed using flow cytometry-based cytotoxicity assays. Switchable CAR-T cells and target cells were incubated at a 2:1 E:T ratio for 48 h at different TM concentrations. Target cells were quantified, and cell lysis rate was calculated by normalizing the cell count for each sample to that of a control sample inoculated only with tumor cells. Data were fitted to sigmoid curves using a four-parameter model with variable slope. The calculated EC50 value was interpreted as a representative numerical value of the TM's potency against these tumor cells.

[0347] Figure 6This study illustrates cell lysis in primary human T cells edited with gene knockout of a switchable CAR (Auto RevCAR-T) or additionally via CRISPR / Cas-mediated TRAC locus (Allo RevCAR-T), and in the presence of different concentrations of the anti-CD123 targeting module (R-TM123). Modified T cells were co-incubated with CD123-expressing AML cell lines MV4-11 or OCI-AML3 in the presence of different concentrations of R-TM123 (i.e., antibody concentrations). OCI-AML3 cells were pre-stained with the cytokine eFlour670. The number of surviving eFlour670-positive AML cells was determined by flow cytometry after 48 h. Cell lysis rate was calculated by normalizing the number of surviving AML cells to a control group containing only AML cells. The resulting dose-response curves, EC50 values, and Hill slope are shown in the figures. Nonspecific lysis was measured in the absence of R-TM123. In this short-term assay, the EC50 values ​​for autologous RevCAR-T cells and allogeneic RevCAR-T cells were comparable. Therefore, the lytic activity of RevCAR-T cells was not affected by TRAC editing. Furthermore, nonspecific lysis of MV4-11 cells was eliminated by performing TRAC editing on allogeneic RevCAR-T cells.

[0348] The efficacy of adoptive immunotherapies, including CAR-T, requires the persistence of metastatic T cells in vivo. This is particularly critical for switchable CAR-T platforms such as RevCAR, as long-term persistence allows for reactivation after pausing administration of soluble adaptor molecules. Certain combinations of surface markers are associated with clinically superior T cell phenotypes, and enriching these phenotypes in clinical T cell products can improve antitumor efficacy and overall clinical outcomes (Mahnke et al., 2013).

[0349] Figure 7Phenotypic analysis of RevCAR-T products manufactured using a clinical-scale, GMP-compliant process is presented. Frozen samples of the formulated final drug product were thawed and stained against surface markers CD4, CD28, CD45RA, CD45RO, and CD197 (CCR7). Human T cell subsets were classified according to the method of Mahnke et al. (Mahnke et al., 2013): Tcm, central memory T cells CD28+CD197+; Ttm, transitional memory T cells CD28+CD197-; and Tem, effector memory T cells CD28-CD197-. Selective enrichment of CD28 and CC chemokine receptor type 7 (CCR7, CD197) double-positive T cells was observed; these T cells were classified as central memory T cells (Tcm). In addition to the major Tcm phenotype found in the product, significantly smaller numbers of transitional memory T cells (Ttm) and memory effector T cells (Tem) were also detected in the RevCAR product. Figure 7 Most T cells (>90%) simultaneously express two markers, CD45RO and CD45RA, on their surface, a hallmark of T cells undergoing cell division (LaSalle and Hafler, 1991). Studies have shown that cell products containing a higher proportion of CD28-positive and / or CCR7-positive T cells are associated with objective tumor response in non-human primate models and patients (see Busch et al., 2016 and Gattinoni et al., 2017).

[0350] R-TM123 dose-dependent activation of Allo-RevCAR-T

[0351] The experiments described in the following sections all used clinical-scale Allo-RevCAR-T. The high-affinity receptor for IL-2, CD25 (IL-2 receptor α, IL-2Rα), is expressed in human T cells and becomes detectable on the cell surface upon stimulation by the endogenous TCR complex (Kmieciak et al., 2009). IL-2Rα regulates the T cell proliferative response and is an indicator of the intensity of TCR stimulation (Shatrova et al., 2016).

[0352] Stimulation of RevCAR by R-TM123 (SEQ ID No. 21) is similar to the activation process induced by endogenous TCR. The difference is that in the artificial receptor, the activation signal from the immune receptor tyrosine-based activation motif (ITAM) of the CD3ζ region is accompanied by a co-stimulatory signal from the CD28 signaling chain (Cartellieri et al., 2016), and this activation process can be tracked by monitoring the upregulation of CD25.

[0353] Figure 8 This figure shows the expression of CD25 on the surface of Allo-RevCAR-T cells under R-TM123-mediated activation. Allo-RevCAR-T batches were co-cultured with CD123-expressing AML cell lines MOLM-13, MV4-11, and OCI-AML3 for 48 h in the presence of different concentrations of R-TM123. Cell samples were prepared, and the surface expression of CD2, RevCAR, CD4, CD8, and CD25 was analyzed by flow cytometry. All samples were pre-gated to CD2+ / RevCAR+ cells. The figures show the frequencies of CD25+ Allo-RevCAR-T cells for CD4+ and CD8+ cells, respectively. The figures also present data from technically replicate samples co-cultured from four clinical-scale batches of healthy donor material, after staining.

[0354] Therefore, in the presence of target cells expressing CD123, the surface expression of CD25 on Allo-RevCAR-T cells was detected to assess their response to R-TM123-mediated stimulation. The frequency of RevCAR-T cells expressing CD25 depended on the dose of R-TM123 ( Figure 8 After Allo-RevCAR-T cells are cross-linked with target cells via R-TM123, RevCAR-expressing CD4+ and CD8+ T cells are activated. Figure 8 After 48 hours, the two subgroups showed similar response strength to the R-TM123 dose. Figure 8 (and Table 2).

[0355] Table 2. Half-maximal dose (EC5) of R-TM123 required for CD25 expression on Allo-RevCAR-T cells 50 ).according to Figure 1 The dose-response curves shown indicate that the median maximal dose (MCD) of CD25-expressed R-TM123 was determined and listed. 50 The data points were fitted using GraphPadprism 9 software. A four-parameter nonlinear regression was employed.

[0356]

[0357] Dose-dependent cytotoxic response of R-TM123 to CD123 leukemia cell lines

[0358] To assess dose-dependent target cell lysis, four clinical-scale batches of Allo-RevCAR-T were used in a cytotoxicity assay. Cytotoxic responses of the four Allo-RevCAR-T batches to three AML cell lines were analyzed at escalating R-TM123 concentrations. Figure 9The dose-response curves of R-TM123 on MOLM-13, OCI-AML3 and MV4-11 cell lines are shown.

[0359] Figure 9 The specific cleavage of the AML cell line MOLM-13 by R-TM123-redirected Allo-RevCAR-T was demonstrated. The CD123-expressing AML cell lines MOLM-13, MV4-11, and OCI-AML3 were labeled with the cytokine eFluor670 and subsequently, in the absence or presence of R-TM123, were subjected to cleavage with 2 × 10⁻⁶ cells from four clinical-scale batches at an E:T ratio of 1:1. 5 Allo-RevCAR-T cells were co-cultured. After 48 hours of co-culture, the number of viable target cells and specific lysis were determined by flow cytometry. The figure shows the mean ± SD values ​​of three technical replicates and the dose-response curves obtained for four clinical-scale Allo-RevCAR-T batches from independent donors. Data points were fitted using a four-parameter nonlinear regression using GraphPad9 software, and the half-maximum dose of R-TM123 is listed in Table 3.

[0360] MOLM-13 cells were derived from the peripheral blood of a patient with relapsed acute monocytic leukemia (FAB M5a), which evolved from myelodysplastic syndrome (Matsuo et al., 1997). The OCI-AML3 cell line was obtained from a patient with AML (FABM4) and carries an NPM1 mutation (type A) and abnormal cytoplasmic translocation of nucleophosphoproteins, an immunocytological marker of NPM1-mutant AML (Quentmeier et al., 2005). Furthermore, it contains the R882C DNMT3A mutation (Tiacci et al., 2012). Therefore, these two cell lines represent major AML subtypes and will be included in upcoming clinical studies. The MV4-11 cell line was originally derived from a pediatric acute monocytic leukemia case and has also been described as expressing CD123 (Mani et al., 2018).

[0361] Allo-RevCAR-T cells induced target cell lysis in all three target cell lines, and lysis was strictly R-TM123-dependent. Half-maximal lysis (EC50) was observed in all three target cell lines. 50 The cleavage levels were in the single-digit picomoles range (Table 3). For all AML cell lines, the four clinical-scale batches of Allo-RevCAR-T showed similar half-maximum lysis levels, reaching the upper limit of lysis (i.e., 100% target cell lysis) at approximately 1 nM.

[0362] Table 3. Half-maximal dose (EC50) of R-TM123 required for lysis of Allo-RevCAR-T-mediated AML target cell lines MOLM-13, MV4-11, and OCI-AML3 in vitro. 50 ).

[0363]

[0364] R-TM123 dose-dependent cell reprogramming of Allo-RevCAR-T cells targeting CD123-expressing leukemia cell lines Cytokine release

[0365] Due to the involvement of the TCR, T cells are activated and release a large number of cytokines. These cytokines have effector, stimulatory, regulatory, chemotactic, and inflammatory functions. Similarly, CAR-engineered T cells also release cytokines when stimulated through their artificial receptors (Rossi et al., 2018).

[0366] Co-culture assays were used to characterize the cytokine release potential of Allo-RevCAR-T cells. For this purpose, Allo-RevCAR-T cells from the same four clinical-scale batches used in the specific target cell lysis and T cell activation studies described in the preceding sections were analyzed. Cells were thawed and co-cultured with MOLM-13AML cells in the presence of R-TM123 for 48 h, and the release of effector cytokines from the cell culture supernatant was quantified using the MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi, Germany).

[0367] Qualitatively, very similar cytokine release profiles were observed across different Allo-RevCAR-T clinical-scale batches, showing significant release of effector cytokines (granzyme B and perforin), as well as elevated levels of pro-inflammatory cytokines (such as GM-CSF, IFN-γ, TNF-α) and IL-2. Figure 10 The anti-inflammatory and regulatory cytokine IL-4 was detected in only two Allo-RevCAR-T batches. In terms of quantification, the absolute amounts of individual cytokines showed differences depending on the donor or product. Furthermore, the half-maximal cytokine release (EC50) of R-TM123 was determined from the sigmoid dose-response curve via nonlinear regression. 50 (See Table 4). EC 50The value can be used as a direct measure of cytokine release kinetics and can be compared with other effector functions, such as target cell lysis or T cell activation. Generally, differences in half-maximal cytokine release among cytokines are more significant than differences between Allo-RevCAR-T cell products. Of particular note is that the effector cytokines granzyme B and perforin were secreted at low R-TM123 concentrations and were associated with target cell lysis (EC50 of granzyme B / perforin). 50 The cytokine release is 4-12 pM, while lysis is 2-6 pM. Pro-inflammatory cytokines only exhibit half-maximal cytokine secretion at much higher doses of R-TM123, such as GM-CSF at approximately 35-40 pM, IFN-γ at approximately 20-25 pM, or TNF-α at approximately 50-80 pM. In summary, Allo-RevCAR-T clinical-scale products exhibit typical T-cell cytokine release characteristics. The amount of cytokine released varies quantitatively between different cytokines and between different T-cell donors or products. Cytokine release is associated with other effector functions of Allo-RevCAR-T tested (i.e., activation and target cell lysis), but higher doses of R-TM123 are required to achieve the corresponding levels.

[0368] Figure 10 The study shows the cytokines and effector molecules released by Allo-RevCAR-T redirected by R-TM123 against the AML cell line MOLM-13.

[0369] In the presence of R-TM123, Allo-RevCAR-T cells, produced from four healthy donors using a clinical-scale process, were co-incubated with the CD123-expressing AML cell line MOLM-13 at a specified concentration and a 1:1 effector-to-target cell ratio. After 48 h, the T cell co-culture supernatant was harvested, technical replicates were combined, and analyzed using flow cytometry-based multiplex assays (MACSPlex Cytotoxic T / NK Cell Kit, Miltenyi, Germany). The corresponding dose-response curves are shown in the figure. Using GraphPad Prism 9 software, sigmoid data points were fitted using four-parameter logistic regression, and half-maximal cytokine release (EC50) was measured. 50 (Summarized in Table 4).

[0370] Table 4. Half-maximal release of cytokines and effector molecules by R-TM123-redirected Allo-RevCAR-T against the AML cell line MOLM-13. (nd = not detected).

[0371]

[0372] Allo-RevCAR-T induces dose-dependent lysis of primary AML cells by R-TM123.

[0373] In a flow cytometry-based cytotoxicity assay, the ability of clinical-scale Allo-RevCAR-T batches to lyse primary patient AML material was analyzed. Primary AML cells (AML1, AML3, AML4, AML5) from four AML patients were combined with three Allo-RevCAR-T batches (i.e., a total of 12 primary AML / Allo-RevCAR-T pairs) for cytotoxicity assays. On the day the assay was initiated, patient-derived AML cells were thawed, washed, and the expression of markers CD45, CD14, HLA-DR, CD33, and CD34 was characterized by flow cytometry to confirm the expression of the target CD123 (data not shown). Allo-RevCAR-T batches were co-cultured with primary AML cells at a 1:2 effector-to-target cell ratio in the presence of R-TM123. After 48 hours of co-culture, the number of surviving AML cells was determined by flow cytometry staining. Figure 11 ).

[0374] Figure 11 The cytotoxic response of R-TM123-redirected Allo-RevCAR-T cells against primary leukemia cells from AML patients was demonstrated. Allo-RevCAR-T cells, derived from healthy donors and manufactured using a clinical-scale process, were thawed and reacted with 1.25–1.5 × 10⁻⁶ cells. 4 Primary AML cells were co-cultured at a 1:2 effector-to-target ratio within a specified R-TM123 concentration range. IMDM supplemented with 5% FBS, 5 μM β-mercaptoethanol, 1% penicillin / streptomycin, 100 ng / mL stem cell factor (SCF), 10 ng / mL IL-3, 10 ng / mL thrombopoietin (TPO), and 10 ng / mL Fms-associated tyrosine kinase 3 ligand (FLT-3L) was used as the culture medium. After 48 h, the number of surviving AML cells was determined by flow cytometry. The dose-response curve was calculated using GraphPad Prism 9 software by performing a four-parameter nonlinear fit on log(agonist) versus response with a variable slope. Table 5 summarizes the calculated EC50 values ​​for target cell lysis.

[0375] Table 5. Efficacy of R-TM123 on primary AML target cells in vitro lysate EC 50 Summary of calculated values.

[0376]

[0377]

[0378] R-TM123 single intravenous administration in mice

[0379] In NOD.Cg-Prkdc scid Il2rg tm1Wjl The in vivo pharmacokinetic characteristics of R-TM123 were investigated in an immunocompromised mouse model (NSG). R-TM123, obtained from validation studies, was administered intravenously (IV) to NSG mice at a single dose of 1000 or 3000 ng / g. Blood was collected via retroorbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300, and 360 minutes post-injection. After plasma separation, the concentration of R-TM123 was measured using a specific sandwich ELISA assay employing CD123-Fc as a capture agent and an anti-polyhis-tagged monoclonal antibody conjugated to horseradish peroxidase (HRP) to detect bound R-TM123. Capture with CD123-Fc indicated the integrity of the anti-CD123 domain, while detection of the polyhis-tagged antibody indicated the presence of a second RCE-specific scFv, located at the N-terminus of the polyhis-tagged antibody. Quantification of the test sample was achieved by comparing it with a titrated dose of R-TM123 standard. The limit of detection for the sandwich ELISA assay was determined to be 0.6 ng / mL assay concentration.

[0380] To determine plasma half-life, non-compartmental model analysis (NCA) and bicompartmental model analysis (2CA) were performed on plasma data following intravenous bolus injection using PkSolver 2.0 software (Zhang et al., 2010). The 2CA data were then processed via W = 1 / C obs 2 Further weighting is applied to focus the fitted model on the final elimination period T. 1 / 2 -β. Figure 12 The results of the experiments are presented in graphs, and Table 6 summarizes the key pharmacokinetic data.

[0381] Figure 12The in vivo pharmacokinetics of R-TM123 in NSG mice are shown. Following administration of R-TM123, plasma concentrations of R-TM123 in peripheral blood samples were analyzed using non-compartmental model analysis (NCA) and two-compartmental model analysis (2CA), and the corresponding graphs are presented. Mice (n=5) were intravenously injected with 1000 or 3000 ng of R-TM123 per gram of body weight. Peripheral blood samples were collected via retroorbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300, and 360 minutes after intravenous injection. NCA and 2CA analyses of R-TM123 concentrations as determined by ELISA were performed using PkSolver 2.0 software (Zhang et al., 2010). Each data point shows the mean ± standard deviation of samples obtained from each of the five mice.

[0382] At a dose of 1000 ng / g body weight, the area under the curve (AUC) was determined to be 255603.5 (NCA) and 220241.7 ng / mL*min (2CA), and at a dose of 3000 ng / g body weight, the AUC was determined to be 644179.4 (NCA) and 555559.9 ng / mL*min (2CA). At a dose of 1000 ng / g body weight, the elimination half-life (T0) of R-TM123 was... 1 / 2 The calculated values ​​were 36.5 min (NCA) and 38.0 min (2CA), and at a dose of 3000 ng / g body weight, the elimination half-life (T) of R-TM123 was... 1 / 2 The calculated values ​​were 47.8 min (NCA) and 45.3 min (2CA). The obtained T... 1 / 2 This is consistent with values ​​reported in the literature for similar scFv constructs (Hutt et al., 2012), and the plasma concentration of R-TM123 was highest at 15 minutes after intravenous injection (i.e., at the earliest time point measured). max The result was as expected. The maximum plasma concentration (C) was detected 15 minutes after injection. max The half-life was measured at 4682.6 ng / g body weight and 11522.7 ng / mL at 1000 ng / g body weight and 3000 ng / g body weight, respectively. The observed short half-life suggests that R-TM123 should be delivered by continuous infusion, as is the case with bispecific T-cell adaptors with relatively short half-lives (Chichili et al., 2015; Hijazi et al., 2018).

[0383] Figure 6 In vivo pharmacokinetics of R-TM123 in NSG mice. The results of the above experiments are summarized below.

[0384]

[0385] Results obtained at 1000 ng / g body weight at 255, 270, 300, and 360 min, or at 3000 ng / g body weight at 360 min, were below the lower limit of quantitation (LLoQ) by 0.67–1.42 ng / mL and were not included in further analysis. For analysis, non-compartmental analysis (NCA) and two-compartmental model analysis (2CA) were applied to plasma data following intravenous bolus administration using PkSolver 2.0 software (Zhang et al., 2010). The observed differences between the two doses are likely due to technical differences in sampling and / or ELISA performance, particularly at lower R-TM123 concentrations. Because plasma levels of R-TM123 are typically low, the LLoQ was reached more quickly at the 1000 ng / g body weight dose (210 min) than at the 3000 ng / g body weight dose (300 min). The plasma half-life is likely underestimated due to the smaller number of valid measurement points for the terminal elimination period. Therefore, at the terminal stage at a dose of 3000 ng / g body weight, the estimates of 47.8 min (NCA model) and 45.3 min (2CA model) calculated based on six plasma half-life measurement points above LLoQ reflect a more reliable dataset.

[0386] In vivo efficacy of Allo-RevCAR-T in different CDX AML models

[0387] In an extramedullary disease AML-CDX model, the in vivo efficacy of R-TM123-redirected Allo-RevCAR-T cells was confirmed using fluorescence-based readings. In this model, on day 0, MOLM-13 cells expressing mCherry, alone or in combination with Allo-RevCAR-T cells, were subcutaneously injected into the flank of NSG mice. R-TM123 was administered daily around the tumor at a dose specified per gram of body weight for four cycles (each cycle being five days). Figure 13 Based on in vivo fluorescence imaging, a sustained antitumor response was observed against MOLM-13AML cells.

[0388] Figure 13 This demonstrates the leukemia elimination effect of R-TM123-redirected Allo-RevCAR-T against extramedullary leukemia (AML). 1×10⁻⁶ 6 MV4-11 cells expressing mCherry, alone or in combination with 5 × 10⁶ cells. 5Allo-RevCAR-T cell combinations from clinical-scale trials were subcutaneously injected into NSG mice, and tumor growth was monitored using optical imaging. Subsequently, mice were injected peritumorally with R-TM123 at a dose indicated per gram of body weight for four cycles, each cycle consisting of five consecutive days of injection, with a two-day interval between cycles. Tumor signal percentages were referenced to the initial measurements (geometric mean, t0) for the corresponding groups. Statistical significance was assessed using Dunn's multi-comparison test via two-way ANOVA. Results showed that all treatment groups had p-values ​​less than 0.05 compared to the group receiving both Allo-RevCAR-T cells and tumor cells without R-TM123.

[0389] Compared to unedited RevCAR-T products, Allo-RevCAR-T exhibits enhanced RevCAR transduction efficiency.

[0390] T cells were isolated from healthy human donors after CD4 and CD8 positivity selection and cultured in 6-well Grex plates (Wilson Wolf, Saint Paul, MN, US). On day 2 post-polyclonal activation, Allo-RevCAR-T cells and unedited RevCAR-T cells were transduced by adding RevCAR-containing viral supernatant at an infection fold-over-infection (MOI) of 2. Before and after retroviral transduction, Allo-RevCAR-T cells underwent gene editing to achieve TRAC, HLA-A, and CIITA gene knockout by adding LNPs containing Cas9 mRNA and the corresponding sgRNA. The genetically engineered T cells were expanded on day 9 post-activation, and RevCAR surface expression was analyzed by flow cytometry. For this purpose, the monoclonal antibody anti-5B9-AF647 was used to identify the functional binding site of RevCAR.

[0391] RevCAR-transduced T cells showed an average transduction efficiency of only 36.7% ± 17.4%, while T cells treated with LNP for TRAC, HLA-A, and CIITA gene knockout had a RevCAR-positive cell rate of 57.9% ± 16.5%. Figure 14 The results showed that modifying T cells with LNPs containing sgRNA / mRNA could enhance RevCAR transduction efficiency.

[0392] Figure 14The proportion of RevCAR-positive T cells in Allo-RevCAR-T is shown compared to the unedited RevCAR-T (Auto-RevCAR-T) product. Data presented are the mean ± SD of RevCAR-T products from 15 independent donors. Statistical significance was calculated using paired t-tests. p < 0.0001 = ****.

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[0430] List of reference numerals

[0431] 1. First structural domain: Tag-associative domain or tag

[0432] 2. Second domain: extracellular hinge and transmembrane domain

[0433] 3. Third structural domain: Signal transduction structural domain

[0434] 4. Optional fourth domain: short peptide linker

Claims

1. An engineered human T cell, comprising: i. Reducing or eliminating the surface expression of endogenous T cell receptor α chain by genetic modification of the T cell receptor α chain gene, ii. by genetically modifying the HLA-A gene, reducing or eliminating the surface expression of HLA-A relative to unmodified T cells, iii. reducing or eliminating the surface expression of HLA class II by genetic modification of the CIITA gene, and iv. a reverse universal chimeric antigen cell surface receptor, the reverse universal chimeric antigen cell surface receptor comprising: Label, The extracellular hinge and transmembrane domain, and Signal transduction domain.

2. The engineered human T cell of claim 1, wherein the tag is a peptide epitope tag.

3. The engineered human T cells according to claim 2, wherein the peptide epitope tag is a myc-tag, a His-tag, a short linear peptide sequence from the yeast transcription factor GCN4, a leucine zipper sequence or from human nucleoprotein, or a short linear peptide sequence from human La protein.

4. The engineered human T cell according to any one of claims 1 to 3, wherein the extracellular hinge and transmembrane domain are selected from portions of human CD28 molecules, CD8a chains, NK cell receptors, or antibody constant regions, mutants thereof, or combinations thereof.

5. The engineered human T cell according to any one of claims 1 to 4, wherein the signal transduction domain is selected from the group consisting of the cytoplasmic regions of the following: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptors, mutants thereof, and combinations thereof.

6. The engineered human T cell according to any one of claims 1 to 5, wherein the T cell is homozygous for both HLA-B and HLA-C genotypes.

7. A pharmaceutical composition comprising the engineered human T cells according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, or for treating cancer, infectious diseases or autoimmune diseases.

9. A kit, comprising: a) Engineered human T cells, comprising: i. Reducing or eliminating the surface expression of endogenous T cell receptor α chain by genetic modification of the T cell receptor α chain gene, ii. by genetically modifying the HLA-A gene, reducing or eliminating the surface expression of HLA-A relative to unmodified T cells, iii. Reducing or eliminating the surface expression of HLA class II by genetic modification of the CIITA gene, and iv. A reverse universal chimeric antigen cell surface receptor comprising: Label, The extracellular hinge and transmembrane domain, and signal transduction domain, and b) a targeting module, or a nucleic acid, a vector or a cell encoding the targeting module, wherein the targeting module comprises a tag binding domain and at least one target cell binding domain, The tag binding domain of the targeting module binds to the tag of the reverse universal chimeric antigen cell surface receptor.

10. The kit of claim 9, wherein the at least one target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and mutants, tumor necrosis factor receptor superfamily members, tight junction proteins, ephrins, ephrin receptors, fucosyltransferases, prostate-specific antigen, embryonic antigens, vascular endothelial growth factor family members, EpCAM, AF P, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligand of the NKG2D receptor, members of the epithelial cell glycoprotein family, disialylganglioside, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus proteins, cancer-testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans.

11. The kit according to claim 9 or 10, wherein the kit further comprises at least one additional targeting module, or a nucleic acid, vector or cell encoding the additional targeting module, wherein the at least one additional targeting module comprises at least one target cell binding domain and a tag binding domain, Wherein the at least one target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that can bind to the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150 CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and mutants, tumor necrosis factor receptor superfamily members, tight junction proteins, ephrins, ephrin receptors, fucosyltransferases, prostate-specific antigen, embryonic antigens, vascular endothelial growth factor family members, EpCAM, AFP, intercellular adhesion molecule family members, C-type lectins, integrins, mucin family members, FSHR, HMW- MAA, FBP, folate receptor, somatostatin receptor, ligands of NKG2D receptor, members of the epithelial cell glycoprotein family, disialogangliosides, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus proteins, cancer-testis antigen, fibroblast activation protein, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans.

12. The kit according to any one of claims 9 to 11, wherein the engineered human T cells and / or the targeting module are in the form of a pharmaceutical composition.

13. The kit according to any one of claims 9 to 12, for use in a method of stimulating a chimeric antigen receptor-mediated immune response in a mammal, or for use in treating cancer, infectious diseases or autoimmune diseases.

14. A kit, comprising: a) A CRISPR system comprising: Nucleic acid targeting a region of the endogenous T cell receptor alpha chain (TRAC) gene, Targeting nucleic acid in a region of the endogenous HLA-A gene, A nucleic acid targeting a region of the endogenous CIITA gene, and b) a nucleic acid or a vector containing a nucleic acid, wherein the nucleic acid encodes a reverse universal chimeric antigen cell surface receptor, wherein the reverse universal chimeric antigen cell surface receptor comprises: Label, The extracellular hinge and transmembrane domain, and signal transduction domain, and c) a targeting module, or a nucleic acid, a vector or a cell encoding the targeting module, wherein the targeting module comprises a tag binding domain and at least one target cell binding domain, The tag binding domain of the targeting module binds to the tag of the reverse universal chimeric antigen cell surface receptor.

15. The kit according to claim 14, wherein the CRISPR system comprises CRISPR associated protein 9 mRNA and nucleic acid targeting TRAC, HLA-A or CIITA, and the CRISPR associated protein 9 mRNA and nucleic acid are both encapsulated in lipid nanoparticles.

16. The kit according to claim 15, wherein the nucleic acid targeting TRAC comprises or encodes the sequence shown in SEQ ID NO.1, and / or the nucleic acid targeting HLA-A comprises or encodes the sequence shown in SEQ ID NO.2, and / or the nucleic acid targeting CIITA comprises or encodes the sequence shown in SEQ ID NO.

3.

17. A method for preparing engineered human T cells expressing a reverse universal chimeric antigen cell surface receptor, the method comprising: 1) Provide human T cells, 2) introducing into human T cells by transfection a CRISPR system comprising a nucleic acid targeting an endogenous T cell receptor α chain region in the T cells, 3) introducing a CRISPR system into the human T cells by transfection, wherein the CRISPR system comprises a nucleic acid targeting the HLA-A region in the T cells, and 4) introducing a CRISPR system into the human T cells by transfection, wherein the CRISPR system comprises a nucleic acid targeting the CIITA region in the T cells, thereby preparing CRISPR-modified T cells, and 5) introducing into the human T cells by transduction a nucleic acid encoding a reverse universal chimeric antigen cell surface receptor, or a vector comprising the aforementioned nucleic acid, wherein the reverse universal chimeric antigen cell surface receptor comprises: Label, The extracellular hinge and transmembrane domain, and Signal transduction domain.

18. The method of claim 17, wherein the T cells are homozygous for both HLA-B and HLA-C genotypes.

19. The method of claim 18, wherein the method further comprises matching the HLA-B and HLA-C alleles of the engineered human T cells to a patient.

20. The method of any one of claims 17 to 19, wherein the CRISPR system comprises a lipid nucleic acid assembly composition comprising an endonuclease or an mRNA encoding an endonuclease, and a site-specific single-stranded guide RNA.

21. The method of claim 20, wherein the endonuclease is CRISPR-associated enzyme 9.

22. The method according to claim 20 or 21, wherein the site-specific single-stranded guide RNA in step (2) comprises the nucleotide sequence shown in SEQ ID NO.1, and / or the site-specific single-stranded guide RNA in step (3) comprises the nucleotide sequence shown in SEQ ID NO.2, and / or the site-specific single-stranded guide RNA in step (4) comprises the nucleotide sequence shown in SEQ ID NO.

3.

23. The method of any one of claims 20 to 22, wherein the lipid nucleic acid assembly composition comprises lipid nanoparticles.

24. The method of claim 23, wherein the lipid nanoparticles are a mixture of cationic lipids and / or ionizable lipids and helper lipids.

25. The method of claim 23 or 24, wherein the lipid nanoparticles are a mixture of ionizable lipids, helper lipids and PEG-DMG.

26. The method according to any one of claims 17 to 25, wherein the introduction of the nucleic acid encoding the reverse universal chimeric antigen cell surface receptor or the vector comprising the nucleic acid according to step (5) is introduced by adding retroviral, lentiviral or adenoviral vector particles.

27. A method for treating cancer, infectious disease or autoimmune disease, comprising the following steps: a) administering an effective amount of a targeting module or a nucleic acid or a pharmaceutical composition comprising the targeting module to a mammal, preferably a human, suffering from cancer, an infectious disease or an autoimmune disease, wherein the targeting module comprises a tag binding domain and at least one target cell binding domain, and b) administering to the mammal an effective amount of the engineered human T cells according to any one of claims 1 to 6, or a pharmaceutical composition comprising said engineered human T cells, wherein the tag binding domain of the targeting module binds to the tag of the reverse universal chimeric antigen cell surface receptor, and wherein the targeting module is administered to the mammal before, simultaneously with, or after administration of the engineered human T cells.

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